NZ615328B2 - Fusion proteins and combination vaccines comprising haemophilus influenzae protein e and pilin a - Google Patents

Abstract

Disclosed is a fusion protein of formula I: (X)m - (R1)n - A - (Y)o - B - (Z)p (formula I) wherein: X is a signal peptide or MHHHHHH; m is 0 or 1; R1 is an amino acid; n is 0, 1, 2, 3, 4, 5 or 6; A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, or PilA from Haemophilus influenzae or an immunogenic fragment thereof; Y is selected from the group consisting of GG, SG, SS, GGG and (G)h wherein h is 4, 5, 6, 7, 8,9, or 10; o is 0 or 1; B is PilA from Haemophilus influenzae or an immunogenic fragment thereof, or Protein E from Haemophilus influenzae or an immunogenic fragment thereof; Z is GGHHHHHH (SEQ 10 NO.3); and p is 0 or 1, wherein when A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, B is not Protein E from Haemophilus influenzae or an immunogenic fragment thereof; and wherein when A is PilA from Haemophilus influenzae or an immunogenic fragment thereof, B is not PilA from Haemophilus influenzae or an immunogenic fragment thereof. Also disclosed is an immunogenic composition comprising isolated Protein E from H. influenzae and isolated PilA from H. influenzae ophilus influenzae or an immunogenic fragment thereof; Y is selected from the group consisting of GG, SG, SS, GGG and (G)h wherein h is 4, 5, 6, 7, 8,9, or 10; o is 0 or 1; B is PilA from Haemophilus influenzae or an immunogenic fragment thereof, or Protein E from Haemophilus influenzae or an immunogenic fragment thereof; Z is GGHHHHHH (SEQ 10 NO.3); and p is 0 or 1, wherein when A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, B is not Protein E from Haemophilus influenzae or an immunogenic fragment thereof; and wherein when A is PilA from Haemophilus influenzae or an immunogenic fragment thereof, B is not PilA from Haemophilus influenzae or an immunogenic fragment thereof. Also disclosed is an immunogenic composition comprising isolated Protein E from H. influenzae and isolated PilA from H. influenzae

Description

/050236 FUSION PROTEINS AND COMBINATION VACCINES COMPRISING HAEMOPHILUS INFLUENZAE PROTEIN E AND PILIN A This application claims priority to United States patent application number 779 filed April 13, 2011 and United States patent application number 61/534012 filed September 13, 2011.

FIELD OF THE ION The present invention relates to itions sing Haemophi/us influenzae (H. influenzae) Protein E and Pilin A. More particularly, the present application relates to fusion proteins and immunogenic compositions comprising Protein E and Pilin A, vaccines comprising such immunogenic compositions and therapeutic uses of the same.

BACKGROUND OF THE INVENTION Protein E (PE) is an outer membrane lipoprotein with adhesive ties. It plays a role in the adhesion/invasion of peable Haemophi/us influenzae (NTHi) to epithelial cells. (J. Immunology 183: 2593-2601 (2009); The Journal of Infectious Diseases 2-531 (2009), Microbes and Infection 10:87-96 (2008)). It is highly conserved in both encapsulated Haemophi/us influenzae and non-typeable H. influenzae and has a conserved epithelial binding . (The Journal of ious Diseases 201:414-419 (2010)). Thirteen different point mutations have been described in different Haemophi/us species when compared with Haemophi/us influenzae Rd as a reference strain. Its expression is observed on both logarithmic growing and stationary phase bacteria. (WO2007/084053).

Protein E is also involved in human complement ance through binding vitronectin.

(Immunology 183: 2593-2601 (2009)). PE, by the binding domain PKRYARSVRQ YKILNCANYH LTQVR (SEQ ID NO. 1, corresponding to amino acids 84-108 of SEQ ID NO. 4), binds vitronectin which is an important inhibitor of the terminal complement pathway. (J.

Immunology 93-2601 (2009)).

Pilin A (PiIA) is likely the major pilin subunit of H. influenzae Type IV Pilus (Tfp) involved in ing motility (Infection and Immunity, 73: 1635-1643 (2005)). NTHi PilA is a conserved adhesin expressed in vivo. It has been shown to be involved in NTHi adherence, colonization and biofilm formation. ular Microbiology 65: 1288-1299 (2007)). peable hilus nzae is an important and common respiratory pathogen that causes otitis media in infants and children. NTHi is, after Streptococcus pneumoniae, the most common cause of acute otitis media in children (J. Immunology 183: 2593-2601 (2009), Pediatrics 113:1451-1465 (2004)). It is an important cause of sinusitis in children and adults.

(Current Infectious Disease Reports 11:177-182 (2009)). It has been associated with increased risk of exacerbations in c obstructive pulmonary disease (COPD) in adults. (Journal of Chronic Obstructive Pulmonary Disease 3:109-115 (2006)). In addition, non-typeable H. influenzae causes community-acquired pneumonia in adults and may cause pneumonia in children in developing countries. (Current Infectious Disease Reports 11:177-182 (2009)).

A need for vaccines for NTHi exists.

BRIEF SUMMARY OF THE INVENTION As a first aspect, the present invention provides fusion proteins of a (I).

(X) m – (R1)n – A – (Y) o – B – (Z)p (formula I) wherein: X is a signal peptide or MHHHHHH (SEQ ID NO. 2); m is 0 or 1; R1 is an amino acid; n is 0, 1, 2, 3, 4, 5 or 6; A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, or PilA from Haemophilus nzae or an immunogenic fragment thereof; Y is selected from the group consisting of GG, SG, SS, GGG and (G)h wherein h is 4, 5, 6, 7, 8, 9, or 10; o is 0 or 1; B is PilA from Haemophilus influenzae or an immunogenic fragment f, or Protein E from Haemophilus influenzae or an immunogenic nt thereof; Z is GGHHHHHH (SEQ ID NO. 3); and p is 0 or 1, n when A is Protein E from hilus nzae or an immunogenic fragment thereof, B is not Protein E from Haemophilus influenzae or an genic fragment thereof; and wherein when A is PilA from Haemophilus influenzae or an immunogenic fragment thereof, B is not PilA from hilus influenzae or an immunogenic fragment thereof.

As a second aspect, the present invention provides immunogenic compositions comprising fusion proteins of formula (I). The composition may r comprise a pharmaceutically acceptable adjuvant. The composition may comprise an excipient.

In a third aspect, the present invention provides a method for the treatment or prevention of a condition or disease caused wholly or in part by Haemophilus influenzae. The method comprises administering to a t in need thereof a therapeutically effective amount of the fusion protein of formula (I). In a related aspect the invention provides for the use of a fusion protein of formula (I) or an immunogenic composition thereof in the manufacture of a medicament for the ent or prevention of a condition or disease caused wholly or in part by Haemophilus influenza.

In a fourth aspect, the present invention provides a method for the treatment or prevention of otitis media. The method comprises administering to a subject in need thereof a therapeutically effective amount of the fusion protein of a (I). In a related aspect the invention provides for the use of a fusion n of formula (I) or an immunogenic composition thereof in the manufacture of a medicament for the treatment or prevention of otitis media In a fifth aspect, the present invention provides a method for the treatment or prevention of exacerbations in chronic obstructive pulmonary disease. The method comprises administering to a subject in need thereof a therapeutically effective amount of the fusion protein of a (I).

In a related aspect the invention provides for the use of a fusion protein of formula (I) or an immunogenic composition thereof in the cture of a medicament for the treatment or prevention of exacerbations in chronic obstructive pulmonary disease. (followed by 3A) In a sixth aspect, the present invention provides a method for the treatment or prevention of pneumonia. The method comprises administering to a t in need thereof a therapeutically effective amount of the fusion n of formula (I). In a related aspect the invention provides for the use of a fusion protein of formula (I) or an genic composition thereof in the manufacture of a medicament for the treatment or prevention of nia.

In a seventh aspect, the present invention provides a pharmaceutical composition comprising a fusion protein of formula (I) for use in the treatment or prevention of a condition or disease caused wholly or in part by Haemophilus influenzae. Pharmaceutical compositions may further comprise a pharmaceutically acceptable adjuvant.

In an eighth aspect, the present invention es nucleic acids encoding the proteins of the invention.

In a ninth , the present invention provides a process of producing nucleic acids of the invention.

Further aspects of the present invention are described in the detailed description of particular embodiments, examples and claims which . (followed by 4) 2012/050236 BRIEF DESCRIPTION OF THE DRAWINGS Figure 1. GE of induced bacterial extracts for fusion protein constructs LVL291, LVL268 and LVL269. Insoluble fraction (l), Soluble fraction (S) and Culture Media fraction (M) were loaded for LVL291, LVL268 and LVL269 before and after ion (ind).

Figure 2. SDS—PAGE and Western blot related to purification extracts for fusion protein ucts LVL291, LVL268 and LVL269. Flow through fraction (Ft), Wash fraction (W) and Elution fraction (E) were loaded for purification of LVL291, LVL268 and LVL269. Anti-his tag was used to probe extracts.

Figure 3. SDS-PAGE of induced bacterial and cation extracts for fusion protein constructs LVL291 and LVL315. Culture Media on (M), Soluble fraction (Sol), lnsoluble fraction (lns), Flow through fraction (Ft), Wash on #1 (W1), Wash fraction #2 (W2) and Elution fraction (E) were loaded for LVL291 and LVL315.

Figure 4. SDS-PAGE of induced bacterial and purification extracts for fusion protein construct LVL312. Culture Media fraction (M), Soluble fraction (Sol), ble fraction (lns), Flow Through fraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction (E) were loaded for LVL312.

Figure 5. SDS-PAGE of induced (1mM and 10uM IPTG) bacterial extracts for fusion protein uct LVL317. Extracts from before (NI) and after induction (ln), Soluble fraction (S), lnsoluble fraction (I).

Figure 6. SDS-PAGE of d (1mM and 10uM IPTG) bacterial extracts for fusion protein construct LVL318. Extracts from before (NI) and after induction (ln), Culture Media fraction (M), Soluble fraction (S), lnsoluble on (I).

Figure 7. CD spectra of PE, PilA and PE-PilA fusion proteins.

Figure 8. Combination of PE and PilA CD spectrum.

Figure 9. PilA thermal denaturation curve.

Figure 10. PE denaturation curve.

Figure 11. PE-PilA fusion protein l ration curve.

Figure 12. Typical SP SepharoseTM Fast Flow chromatogram.

Figure 13. Typical Q SepharoseTM Fast Flow chromatogram.

Figure 14. SDS-PAGE of ln-process samples from purification process of PE-PilA fusion protein.

Figure 15. Western Blot of ln-process samples of purification process from PE-PilA fusion protein. Blot using rabbit polyclonal anti-PE.

Figure 16. Western Blot of ln-process samples of cation process from PE-PilA fusion protein. Blot using rabbit polyclonal anti-Eco/i (BLR).

Figure 17. Thermal transition of PE-PilA fusion protein and PE and PilA ns. Curves: PilA (1), Protein E (Prot E, PE) (2), PE-PilA Purified Bulk not diluted, 737ug/ml (3), and PE-PilA Purified Bulk d at Final Container tration 60ug/ml (4).

Figure 18. Antibody responses against LVL291 PE-PilA fusion n and against monovalent PE and PilA in the Balb/c mouse model.

Figure 19. Effect of PE-PilA fusion protein vaccination on NTHi strain 86-028NP bacterial clearance in mouse nasopharynx.

Figure 20. Effect of PE-PilA fusion protein ation on NTHi strain 3224A bacterial clearance in mouse nasopharynx.

Figure 21. Effect of PilA vaccination on bacterial clearance in mouse nasopharynx. 2012/050236 Figure 22. Effect of PE vaccination on bacterial nce in mouse nasopharynx.

Figure 23. (a) LVL317 PE-PilA fusion protein binding to vitronectin and (b) LVL317 and LVL735 PE-PilA fusion protein bound to vitronectin.

Figure 24. tion of vitronectin binding by polyclonal antibodies against PE-PilA fusion protein.

Figure 25. SDS-PAGE of e fractions of induced bacterial extracts for fusion protein constructs LVL291, , LVL736, LVL737, LVL738, LVL739, LVL740 and pET26b vector (negative control). (a) Experiment 1 (b) Experiment 2 (c) Experiment 3. PE-PilA fusion protein indicated by arrow.

Figure 26. The average band percentage of fusion protein in the soluble fraction from Experiments 1, 2 and 3.

Figure 27. PE and PilA antibody response to LVL317 and LVL735.

Figure 28. Effect of LVL735 and LVL317 vaccination on bacterial clearance in a mouse model of non-typeable Haemophi/us influenzae nasopharyngeal zation.

DETAILED DESCRIPTION OF THE INVENTION Unless othenNise explained or defined , all technical and scientific terms used herein have the same g as commonly understood by one of ordinary skill in the art to which this disclosure belongs. For example, definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, hed by Oxford University Press, 1994 (ISBN 0854287—9); Kendrew et al. (eds.), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 002182-9); and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH hers, Inc., 1995 (ISBN 1569—8).

The singular terms “a, ” uan,” and “the” include plural referents unless context clearly tes othenNise. Similarly, the word “or” is intended to include “and” unless the context clearly indicates otherwise. It is r to be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values, given for nucleic acids or polypeptides are approximate, and are provided for ption. Additionally, numerical limitations given with respect to concentrations or levels of a substance, such as an n may be approximate. Thus, where a concentration is indicated to be (for example) approximately 200 pg, it is intended that the concentration includes values slightly more or slightly less than (“about” or “~”) 200 pg.

Although methods and materials similar or equivalent to those described herein can be used in the practice or g of this disclosure, le methods and materials are described below.

The term “comprises” means “includes”. Thus, unless the context es othenNise, the word “comprises,” and variations such as “comprise” and “comprising” will be understood to imply the inclusion of a stated compound or composition (e.g., nucleic acid, polypeptide, antigen) or step, or group of compounds or steps, but not to the exclusion of any other compounds, composition, steps, or groups thereof. The iation, “9.9.” is derived from the Latin exempli gratia, and is used herein to indicate a non-limiting example. Thus, the abbreviation “9.9.” is synonymous with the term “for example.” In order to facilitate review of the various embodiments of this disclosure, the following explanations of terms are ed. Additional terms and explanations are provided in the context of this disclosure.

A “subject” as used herein is a mammal, including humans, non-human primates, and non-primate s such as members of the rodent genus (including but not limited to mice and rats) and members of the order Lagomorpha (including but not limited to rabbits).

As used herein in E”, “protein E”, “Prot E”, and “PE” mean Protein E from H. influenzae. Protein E may consist of or comprise the amino acid sequence of SEQ ID NO. 4 (VKK T. GT.T.TACSAQI QKAEQNDVKL APPTDVRSGY IIRLVKNVNYY DSfiS WVDW QfiL’Q VHE'DA WNLDKGLYV YPEPKRYARS LNCA NY {LTQVQTD FY33 FWGQGL RAAP {KQKKH TLSLTPDTTL YWAAQ CAN YGEAFSVDKQ as well as sequences with at least or exactly 75%, 77%, 80%, 85%, 90%, 95%, 97%, 99% or 100% identity, over the entire length, to SEQ ID NO. 4. Comparison of 53 sequences of Protein E from Haemophi/us influenzae (Table 1, SEQ ID NO. 5 — SEQ ID NO. 57) demonstrated approximately 77% to approximately 100% identity to n E as set forth in SEQ ID NO. 4. For example, in the amino acid sequence of Protein E, amino acid #20 may be isoleucine (I) or threonine (T); amino acid #23 may be alanine (A) or valine (V); amino acid #24 may be lysine (K) or glutamic acid (E); amino acid #31 may be alanine (A) or threonine (T); amino acid #32 may be e (P) or alanine (A); amino acid #34 may be threonine (T) or alanine (A); amino acid #37 may be arginine (R) or glutamine (Q); amino acid #47 may be valine (V) or alanine (A); amino acid #57 may be tryptophane (W) or may be absent (-); amino acid #70 may be e (A) or threonine (T); amino acid #93 may be glutamine (Q) or absent (-); amino acid #109 may be threonine (T) or isoleucine (I); amino acid #119 may be glycine (G) or serine (S); amino acid #153 may be glutamic acid (E) or lysine (K); amino acid #156 may be serine (S) or leucine (L); amino acid #160 may be lysine (K) or gine (N); amino acid #161 may be lysine (K), isoleucine (I) or absent (-); amino acids #162 - #195 may be absent, or as set forth in SEQ ID NO. 15 (with (-) indicating amino acid #166 is absent) or as set forth in SEQ ID NO. 16; or any combination thereof.

Protein E may consist of or se an amino acid sequence that differs from SEQ ID NO. 4 at any one or more amino acid selected from the group consisting of: amino acid #20, amino acid #23, amino acid #24, amino acid #31, amino acid #32, amino acid #34, amino acid #37, amino acid #47, amino acid #57, amino acid #70, amino acid #93, amino acid #109, amino acid #119, amino acid #153, amino acid #156, amino acid #160, amino acid #161 and amino acids #162-#195, wherein amino acid #20 is threonine (T); amino acid #23 is valine (V); amino acid #24 is lysine (K); amino acid #31 is threonine (T); amino acid #32 is alanine (A); amino acid #34 is alanine (A); amino acid #37 is glutamine (Q); amino acid #47 is e (A); amino acid #57 is absent (-); amino acid #70 is threonine (T); amino acid #93 is absent (-); amino acid #109 is isoleucine (I); amino acid #119 is serine (S); amino acid #153 is lysine (K); amino acid #156 is leucine (L); amino acid #160 is asparagine (N); amino acid #161 is lysine (K) or isoleucine (I); or amino acids #162 - #195 are as set forth in SEQ ID NO. 15 (with (-) indicating amino acid #166 is absent) or as set forth in SEQ ID NO. 16.

Table 1: Protein E amino acid sequences from 53 strains of Haemophi/us influenzae (SEQ ID NO. 5 - SEQ ID NO. 57). - indicates amino acid is absent. m Protein E ce .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.5) RdKWZO .G.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .DQG.YVYPEPKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK _ .YNAAQIICAVYGKAFSVDK< SEQ ID V0.6) 86-028NP CSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK _ .YNAAQIICAVYGEAFSVDK< SEQ ID V0.7) R2846 .G.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK _ 1T.YNAAQIICA_VYGKAFSV < SEQ ID V0.8) R2866 .G.L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK _ 1T.YNAAQIICA_VYGKAFSV < SEQ ID V0.9) 3655 .G.L"ACSAQIQ{AEQNDMK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK _ 1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.10) PittAA .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK _ 1T.YNAAQIICA_VYGEAFSV < SEQ ID V0.11) .G.L"ACSAQIQ{AEQNDMK.A?P"DVRSGYIQLVKNVNYYIDSESI-VDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGKAFSV {(SEQ ID N0.12) .G.L"ACSAQIQ{AEQNDVK.APPTDVRSGYIRLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGKAFSVDK< (SEQ ID N0.13) .L"ACSAQIQKAEQNDVK.APP"DVRSGYIRLVKNVNYYIDSESIWVDNQ {G.YVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICANYGKAFSVDKK SEQ ID No.14) .L"ACSAQTQKAEQNDVK.TPP"DVQSGYVRLVKNVNYYIDSESIWVDNQ PEPKRYARSVRQYKILNCANYHLTQVRIDFYDEFWGQGLRAAPK ?Dr .YNAAQIICANYGKAFSVDKNKKICT—LISLNFIQLLGCREYSIFLQLLLFYC EQ ID No.15) .L"ACSAQIQKAEQNDVKLAPPTDVRSGYIRLVKNVNYYIDSESIWVDNQ {G.YVYPEPKRYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK "PDF .YNAAQIICANYGKAFSVDKKIKKICTLISLNFIQLLGCREYSIFLQLLLFYC EQ ID No.16) .G.L"ACSAQIQ{AEQNDMK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ VYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICANYGKAFSV < SEQ ID No.17) .G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICANYGKAFSVDK< SEQ ID No.18) .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_NYGEAFSVDK< SEQ ID No.19) 038144S1 CSAQTQ<VEQNDVK.TAP"DVRSGFVQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_NYGKAFLVDK< SEQ ID No.20) 810956 .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_NYGEAFSV < SEQ ID No.21) 821246 .G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_NYGKAFSVDK< SEQ ID No.22) 840645 .L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.23) 902550Zl9 .L"ACSAQTQ<VEQNDVK.T?P"DVRSGYVQLVKNVNYYIDSESIWVDNQ PEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.24) A840l77 .L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.25) A860514 .L"ACSAQTQ<VEQNDVK.TAP"DVRSGYVQLVKNANYYIDSESIWVDNQ PEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.26) A950014 .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRIDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.27) 306543X4 .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK<(SEQ ID N0.28) A930105 AQIQ{AEQNDVK.A?PTDVRSGYIRLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK<(SEQ ID N0.29) 901905U AQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.30) A920030 .L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.31) .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ IVHFDAVVV {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSV 27Wll679lN CSAQTQ<VEQNDVK.T?P"DVRSGYVQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGKAFSV < SEQ ID V0.33) .G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.34) .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGEAFSVDK< SEQ ID V0.35) .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGEAFSVDK< SEQ ID V0.36) .G.L"ACSAQTQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ VYPEPKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.37) .G.L"ACSAQTQ<VEQNDVK.TAPADVRSGYVQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK AQIICA_VYGKAFSVDK< SEQ ID V0.38) .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.39) .G.L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYAQSVR-YKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.40) .G.L"ACSAQIQ{AEQNDVK.APP"DVRSGYIQLVKNVNYYIDSESIWVDNQ .D<G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1T.YNAAQIICA_VYGKAFSVDK< SEQ ID V0.41) .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK IICAVYGKAFSVDK< SEQ ID V0.42) .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.43) .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.44) .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQSLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.45) .T."‘ACSAQTQ {VEQNDVK .T E’Pr1DVRSGYVQLVKNVNYYI DSES IWVDNQI {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK 1TVYNAAQIICAVYGKAFSVDK< SEQ ID V0.46) .GT.T."ACSAQTQ {VEQNDVK .T SGYV QLVKNVNYYI DSES IWVDNQI .DKG.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK AQIICAVYGKAFSVDK< SEQ ID V0.47) .L"ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.48) .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.49) .L"ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK<(SEQ ID N0.50) {KII .L"ACSAQTQ{AEQNDVK.TPPTDVRSGYIRLVKNVNYYIDSESIWVDNQ IVHFDAVVV {G.YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSV .."ACSAQIQ{AKQNDVKEA?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.52) AQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.53) . ."ACSAQTQ VK .A .3?" DVRSGYI QLVKNVNYYI DSES IWVDNQI PKQYARSVRQYKILNCANYHLTQIRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK<(SEQ ID N0.54) . ."ACSAQIQ {AKQNDVKEA .3?" DVRSGYI QLVKNVNYYI DSES I .YVYPEPKRYAQSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGEAFSVDK< SEQ ID V0.55) .."ACSAQIQ{AEQNDVK.A?P"DVRSGYIQLVKNVNYYIDSESIWVDNQ .YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.56) .."ACSAQIQ{AEQNDMK.A?P"DVRSGYIQLVKNVNYYIDSESI-VDNQ .YVYPEPKQYARSVRQYKILNCANYHLTQVRTDFYDEFWGQGLRAAPK .YNAAQIICAVYGKAFSVDK< SEQ ID V0.57) n E may be Protein E from H. influenzae strain 3224A, RdKW20, 86-028NP, R2846, R2866, 3655, PittAA, PittEE, PittHH, PittII, R3021, 22.4-21, 3219C, 3185, 3241A, 03814481, 810956, 821246, 840645, 902550Z19, A840177, A860514, A950014, 306543X4, A930105, U, A920030, 3221B, 27W116791N, N218, N163, N162, N107, N91, D211PG, D211PD, , D201PD, D198PG, D198PD, D195PD, D189PG, D189PD, D129CG, D124PG, D124PD, D58PG, D33OD, B8433, B8432, 1714, 1128 or B8430. Protein E may be Protein E as set forth in any of SEQ ID NO. 5 — SEQ ID NO. 57.

Protein E may be a sequence with at least 95% identity, over the entire length, to any of SEQ ID NO. 4 — SEQ ID NO. 57. Protein E may be a sequence with at least 95% identity, over the entire length, to any of the sequences set forth in Table 1, SEQ ID NO. 5 — SEQ ID NO. 57.

Immunogenic fragments of Protein E comprise immunogenic fragments of at least 7, 10, , 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 4. The immunogenic nts may elicit antibodies which can bind SEQ ID NO. 4.

Immunogenic nts of Protein E may comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of any of SEQ ID NO. 4 — SEQ ID NO. 57.

The immunogenic fragments may elicit antibodies which can bind the full length sequence from which the fragment is derived.

Immunogenic fragments of Protein E comprise immunogenic fragments of at least 7, 10, , 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 5 — SEQ ID NO. 57. The immunogenic fragments may elicit antibodies which can bind the full length sequence from which the fragment is derived.

As used herein “PilA” means PiIin A from H. influenzae. PilA may consist of or comprise the protein sequence of SEQ ID NO. 58 (MKLTTQQTLK KGh'TT. *ZTM V A A TAT A BSYQNYT ELLQ ASAPYKADVIfiL‘L LCVYSTNIZTT.L NCTGGKNGIA ADITTAKGYV KSVTTSWGA: TVKGDGTLAN M*'.Y T.QATGN AATGVTWTTT SLFP VTQ) as well as sequences with 80% to 100% identity to SEQ ID NO. 58. For example, PilA may be at least 80%, 85%, 90%, 95%, 97% or 100% identical to SEQ ID NO. 58. Full length comparison of 64 ces of PilA from hi/us influenzae (Table 2, SEQ ID NO. 58 — SEQ ID NO. 121) demonstrated approximately 80% to 100% ty to PilA as set forth in SEQ ID NO. 58. For example, in the amino acid sequence of PilA, amino acid #6 may be glutamine (Q) or leucine (L); amino acid #7 may be glutamine (Q) or threonine (T); amino acid #37 may be ine (Q) or lysine (K); amino acid #44 may be alanine (A) or serine (S); amino acid #57 may be alanine (A) or serine (S); amino acid #67 may be asparagine (N) or glycine (G); amino acid #68 may be glutamic acid (E) or lysine (K); amino acid #69 may be theronine (T) or proline (P); amino acid #71 may be lysine (K), asparagine (N), serine (S) or threonine (T); amino acid #73 may be threonine (T), serine (S) or methionine (M); amino acid #76 may be lysine (K), serine (S) or asparagine (N); amino acid #84 may be threonine (T) or lysine (K); amino acid #86 may be alanine (A) or valine (V); amino acid #91 may be lysine (K) or alanine (A); amino acid #94 may be threonine (T), isoleucine (I) or lysine (K); amino acid #96 may be serine (S) or glutamine (Q); amino acid #97 may be asparagine (N) or serine (8); amino acid #99 may be alanine (A) or glycine (G); amino acid #103 may be alanine (A) or lysine (K); amino acid #109 may be aspartic acid (D), alanine (A) or threonine (T); amino acid #110 may be glycine (G), asparagine (N), or arginine (R); amino acid #112 may be serine (S) or glutamic acid (E); amino acid #114 may be threonine (T) or isoleucine (l); amino acid #116 may be threonine (T) or glutamine (Q); amino acid #118 may be glutamic acid (E), threonine (T), alanine (A), lysine (K) or serine (8); amino acid #121 may be serine (S) or alanine (A); amino acid #122 may be alanine (A) or threonine (T); amino acid #123 may be lysine (K), threonine (T) or alanine (A); amino acid #128 may be lysine (K) or threonine (T); amino acid #135 may be aspartic acid (D) or glutamic acid (E); amino acid #136 may be alanine (A) or threonine (T); amino acid #145 may be glycine (G) or arginine (R); amino acid #149 may be glutamine (Q) or lysine (K); or any ation thereof.

Pil A may consist of or se an amino acid sequence that differs from SEQ ID NO. 58 at any or more amino acid selected from the group consisting of amino acid #6, amino acid #7, amino acid #37, amino acid #44, amino acid #57, amino acid #67, amino acid #68, amino acid #69, amino acid #71, amino acid #73, amino acid #76, amino acid #84, amino acid #86, amino acid #91, amino acid #94, amino acid #96, amino acid #97, amino acid #99, amino acid #103, amino acid #109, amino acid #110, amino acid #112, amino acid #114, amino acid #116, amino acid #118 amino acid, #121, amino acid #122, amino acid #123, amino acid #128, amino acid #135, amino acid #136, amino acid #145 and amino acid #149, wherein amino acid #6 is leucine (L); amino acid #7 is threonine (T); amino acid #37 is lysine (K); amino acid #44 is serine (8); amino acid #57 is serine (8); amino acid #67 is glycine (G); amino acid #68 is lysine (K); amino acid #69 is proline (P); amino acid #71 is lysine (K), serine (S) or threonine (T); amino acid #73 is serine (S) or methionine (M); amino acid #76 is serine (S) or asparagine (N); amino acid #84 is lysine (K); amino acid #86 is valine (V); amino acid #91 is e (A); amino acid #94 is isoleucine (l) or lysine (K); amino acid #96 is glutamine (Q); amino acid #97 is serine (8); amino acid #99 is glycine (G); amino acid #103 is alanine (A); amino acid #109 is aspartic acid (D) or threonine (T); amino acid #110 is e (G) or arginine (R); amino acid #112 is serine (8); amino acid #114 is threonine (T); amino acid #116 is threonine (T); amino acid #118 is ic acid (E), alanine (A), lysine (K) or serine (8); amino acid #121 is serine (8); amino acid #122 is threonine (T); amino acid #123 is lysine (K) or alanine (A); amino acid #128 is lysine (K); amino acid #135 is ic acid (E); amino acid #136 is threonine (T); amino acid #145 is arginine (R); amino acid #149 is lysine (K).

Table 2: Pilin A amino acid sequences from 64 strains of Haemophi/us influenzae (SEQ ID NO. 58—SEQ ID NO. 121). m PilA sequence 86-028NP 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.58) NTHi3219C 1QQTI{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKADVELCVY I KNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGVISYTLTAEGDSAKGVTWK 1DASEFPANFCGSV"Q (SEQ ID No.59) 24A 1QQTI{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY I 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV'EQ (SEQ ID No.60) NTHllZ MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSSCSGGSNGIAADI"TAKGYVASVITQSGGITVKGDGTLANWEYILQAAGNAAAGVTWT T"CKGTDASEFPANFCGSV"Q (SEQ ID No.61) 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.62) NTHi67 MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKSDVELCVY S"GKPSTCSGGSNGIAADI"TVKGYVKSV"TSNGAITVAGNGTLDGWSYTLTAEGDSAKGVTWT DASEFPANFCGSV"Q (SEQ ID No.63) 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.64) 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV'DQ (SEQ ID No.65) MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSELLQASAPYKADVZ 1NCTGGKNGIAADI"TAKGYVKSVTTSNGAITVKGDGTLANMEYILQATGNAATGVTWT 1DASEFPANFCGSV'EQ (SEQ ID No.66) QTL{KGFTLIELMIVIAIIAILATIAIPSYKNYTKKAAVSE.LQASAPYKADVELCVY S"NEI"NCMGGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAAAGVTWT DASEFPANFCGSI"Q (SEQ ID No.67) 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANVIEYILQAKGNATAGVTWT 1DASEFPANFCQSV"K (SEQ ID No.68) .QT.{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1SC"GGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANVIEYILQAKGNAAAGVTWT 1DASEFPANFCGSVTK (SEQ ID No.69) MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSSCSGGSNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANWEYILQASGNAATGVTWT T"CKG"DASEFPANFCGSV"Q (SEQ ID No.70) .QT.{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1SC"GGKNGIAADIKTAKGYVASVITQSGGITVKGNGTLANVIEYILQAKGNAAAGVTWT 1DASEFPANFCGSVTK (SEQ ID No.71) MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQATGNAATGVTWT T"CKG"DASEFPANFCGSV"Q (SEQ ID No.72) MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKSDVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT T"CKGTDASEFPANFCQSV"K (SEQ ID No.73) KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.74) 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT 1EAS.FPANFCGSV"Q (SEQ ID No.75) 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKADVELCVY KNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANVIEYILQAKGNATAGVTWT 1DASEFPANFCQSV"K (SEQ ID No.76) MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQATGNAATGVTWT T"CKG"DASEFPANFCGSV"Q (SEQ ID No.77) MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT T"CKGTDASEFPANFCQSV"K (SEQ ID No.78) 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT 1EAS.FPANFCGSV"Q (SEQ ID No.79) 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY GKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT 1EAS.FPANFCGSV"Q (SEQ ID No.80) MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKSDVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGWSYTLTAEGDSAKGVTWK T"CKGTDASEFPANFCGSV"K (SEQ ID No.81) MKLTTQQT.{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"NI KNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT T"C 1DASEFPANFCGSV"Q (SEQ ID No.82) MKLTTQQT.{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"NEA"KC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANWEYILQASGNAATGVTWT T""C 1DASEFPANFCGSV'EQ (SEQ ID No.83) 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.84) 038144Sl MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAISE.LQASAPYKSDVELCVY STGKPSTCSGGSNGIAADITTAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT TTCKGTDAS. EQ ID No.85) 821246 LIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT 1EAS.FPANFCGSV"Q (SEQ ID No.86) 840645 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.87) Zl9 MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKSDVELCVY S"GKPSTCSGGSNGIAADI"TVKGYVKSV"TSNGAITVAGNGTLDGWSYTLTAEGDSAKGVTWT T"CKGTDASEFPANFCGSV"Q (SEQ ID No.88) A840177 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY KNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV'EQ (SEQ ID No.89) A920030 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1NC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV'EQ (SEQ ID No.90) A950014 MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASA?YKADVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDRMSYTLTAEGDSAKGVTWT T"CKGTDASEFPANFCGSV"Q (SEQ ID No.91) 90l905U MKLTTQQTL{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSSCSGGSNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANWEYILQASGNAATGVTWT T"CKGTDASEFPANFCGSV"Q (SEQ ID No.92) A920029 1QTT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKSDVELCVY 1NC"GGKNGIAADI"TAKGYVASVITQSGGITVKGNGTLTNVIEYILQATGNAATGVTWT 1DASEFPANFCGSI"Q (SEQ ID No.93) A930105 .QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT 1CKG"DASEFPANFCGSVTQ (SEQ ID No.94) 306543X4 MKLTTQQTL<KGFTLIELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY S"GKPSSCSGGSNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT T"CKGTDASEFPANFCGSV"Q (SEQ ID No.95) MKLTTQQF.<KGFTLIELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY S"NI "KC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT Tmc 1Dms CGSV"Q (SEQ ID No.96) 1QQr .(KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY "NC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.97) 1QQT.<KGFTT.IELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY "NC"GGKNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (SEQ ID No.98) MKLTTQQTL<KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQAKGNATAGVTWT T"CKGTDASEFPANFC QSV"K (SEQ ID No.99) MKLTTQQTL<KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQAKGNATAGVTWT T"CKGTDASEFPANFC QSV"K (SEQ ID No.100) "QQT - {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'. 4 .LQASAPYKADVELCVY "NC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DAS EFPANFCGSV"Q (SEQ ID ) MKLTTQQTL<KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADIF1TAKGYVASVKTQSGGITVKGNGTLANVIIEYILQAKGNATAGVTWT T"CKGTDASEFPANFC QSV"K (SEQ ID No.102) MKLTTQQT.<KGFTLIELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY S"NI "KC"GGKNGIAADI"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT T""C 1DAS CGSV"Q (SEQ ID No.103) 1QQT.<KGFTT.IELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 PYKADVELCVY "TNC"GGKNGIAADITTAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT TTCKGTDAS.4FPANFCGSVTQ (SEQ ID NO.IO4) "QQT. {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'._‘4 .LQASAPYKADVELCVY "NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (S EQ ID No.105) "QQT _‘ - {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'. .LQASAPYKADVELCVY "NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIIEYILXATGNAATGVTWT 1DASEFPANFCGSV"Q (S EQ ID NO.IO6) "QQT - {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'. 4 .LQASAPYKADVELCVY "NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (S EQ ID NO.IO7) "QQT _‘ - {KGFTTI'ELMIVIAIIAILAF1IAIPSYQNYTKKAAVS'. .LQASAPYKADVELCVY "NC"GGKNGIAADIF"TAKGYVKSVF1TSNGAITVKGDGTLANVIEYILQATGNAATGVTWT 1DASEFPANFCGSV"Q (S EQ ID NO.IO8) MKLTT.QTL {KGFTTI'ELMIVIAIIAILATIAIPSYQNYTKKAAVSH_‘4 .LQASAPYKADVELCVY S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT T"CKGF1DASEFPANFCGSVTQ (S EQ ID NO.IO9) MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE PYKADVELCVY S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT T"CKG"DASEFPANFCGSVTQ (SEQ ID NO.llO) 1QQT.{KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASAE’YKADVELCVY 1SC"GGKNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGVISYTLTAEGDSAKGVTWK 1DAS.4FPANFCGS\/""Q (SEQ ID NO.lll) KGFTLIETMIVIAIIAILA"IAIPSYQNYTKKAAVSELLQASAE’YKADVELCVY 1SC"GGKNGIAADI"TAKGYVKSV"TSNGAITVAGNGTLDGVISYTLTAEGDSAKGVTWK 1DASEFPANFCGSV"Q (SEQ ID No.112) MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE .LQASAPYKADVELCVY S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT DASEFPANFCGSVTQ (SEQ ID NO.ll3) MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT T"CKG"DASEFPANFCGSVTQ (SEQ ID NO.ll4) MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANWEYILQATGNAATGVTWT Tr 1DASEFPANFCGSVTQ (SEQ ID No.115) 1QQT.{KGFTLIETMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1\IC"GGKNGIAADI"TAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT 1EAS.FPANFCGSV"Q (SEQ ID NO.ll6) .QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY 1GKPSTCSGGNNGIAADIKTAKGYVASVKTQSGGITVKGDGTLANVIEYILQATGNAATGVTWT 1CKG"DAS_JFPANFCGSVTQ (SEQ ID ) MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT T"CKGTDASEFPANFCQSV"K (SEQ ID NO.ll8) MKLTTQQT.{KGFTLIELMIVIAIIAILA"IAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"NI KNGIAADI"TAKGYVKSV"TSNGAITVKGDGTLANVIEYILQASGNAATGVTWT T""C 1DASEFPANFCGSV'EQ (SEQ ID NO.ll9) MKLTT.QTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKAAVSE.LQASAPYKADVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQATGNAATGVTWT T'ECKG'EDASEFPANFCGSV'EQ (SEQ ID No.120) MKLTTQQTL{KGFTLIELMIVIAIIAILATIAIPSYQNYTKKASVSE.LQASAPYKSDVELCVY S"GKPSTCSGGSNGIAADI"TAKGYVASVKTQSGGITVKGNGTLANWEYILQAKGNATAGVTWT T"CKGTDASEFPANFCQSV"K (SEQ ID No.121) PiIA may be PiIA from H. influenzae strain NTHi3219C, NTHi3224A, NTHi12, NTHi44, NTHi67, 1054MEE, E, 1728MEE, E, 1060MEE, RdKW20, 214NP, 1236MEE, 1714MEE, E, 86-028NP, R2846, R2866, 3655, PittAA, PittGG, , R3021, 22.4-21, 3185A, 3221B, 3241A, 03814481, 821246, 840645, 902550Z19, A840177, A920030, 4, 901905U, A920029, A930105, 306543X4, N218, N163, N162, N120, N107, N92, N91, D219PG, D211PG, D211PD, D204CD, D198PG, D198PD, D195PD, D195CD, D189PG, D189PD, D124PG, D124PD, D124CG, D58PG, BS433, BS432, BS430, 1714 or 1128. An amino acid sequence for PilA from H. influenzae strain D204CD is set forth in SEQ ID NO. 106, wherein X at position #116 is either glutamine (Q) or Ieucine (L); ambiguity as to the amino acid at position #116 could be cleared up by cal tion of the second nucleotide encoding amino acid #116, clarifying the PilA sequence for strain D204CD. PilA may be PilA as set forth in any of SEQ ID NO. 58 — SEQ ID NO. 121.

PilA may be a sequence with at least 95% identity, over the entire length, to any of SEQ ID NO. 58 — SEQ ID NO. 121 (as set out in Table 2). lmmunogenic fragments of PilA comprise immunogenic fragments of at least 7, 10, 15, , 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58 — SEQ ID NO. 121. The genic fragments may elicit dies which can bind the full length sequence from which the fragment is derived.

For example, genic nts of PilA comprise immunogenic fragments of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58. The immunogenic nts may elicit antibodies which can bind SEQ ID NO. 58.

Identity between polypeptides may be calculated by various algorithms. For example, the Needle program, from the EMBOSS package (Free software; : The European Molecular Biology Open Software Suite (2000). Trends in Genetics 16(6): 276—277) and the Gap program from the GCG® package (Accelrys Inc.) may be used. This Gap program is an implementation of the Needleman-Wunsch algorithm described in: Needleman, S. B. and Wunsch, C. D. (1970) J. Mol. Biol. 48, 443-453. The BLOSUM62 scoring matrix has been used, and the gap open and extension penalties were respectively 8 and 2.

Looking at the computed alignment, identical residues between two compared sequences can be observed. A percentage of identity can be computed by (1) ating the number of identities divided by the length of the alignment, multiplied by 100 (for example, for the Needle program analysis), (2) calculating the number of identities divided by the length of the longest sequence, multiplied by 100, (3) calculating the number of ties divided by the length of the st sequence, multiplied by 100, or (4) calculating the number of identities divided by the number of aligned residues, multiplied by 100 (a residue is aligned if it is in front of another) (for e, for the Gap program analysis).

As used herein, “adjuvant” means a nd or substance that, when administered to a subject in conjunction with a vaccine, immunotherapeutic, or other antigen- or gen- containing composition, increases or enhances the subject’s immune response to the administered antigen or immunogen (as compared to the immune response that would be obtained in the absence of adjuvant). This is to be guished from “adjuvant therapy”, defined by the National Cancer Institute of the United States Institutes of Health in the context of cancer treatment as additional treatment given after the primary treatment, to lower the risk that the cancer will recur.

Conservative substitutions are well known and are generally set up as the default scoring matrices in sequence ent computer programs. These programs include PAM250 (Dayhoft MO. et al., , “A model of evolutionary changes in proteins”, In “Atlas of Protein sequence and structure” 5(3) MO. Dayhoft (ed.), 345-352), National Biomedical Research Foundation, gton, and Blosum 62 (Steven Henikoft and Jorja G. Henikoft (1992), “Amino acid substitution matrices from protein blocks”), Proc. Natl. Acad. Sci. USA 89 (Biochemistry): 10919. The invention further provides fusion proteins of formula (I) containing vative amino acid substitutions. For example, the fusion proteins of formula (I) may contain a vative substitution of any amino acid from PE or PilA of H. influenzae as described in any of the sequences set forth herein (for example, any PE sequence set forth in SEQ ID NO. 4 — SEQ ID NO. 57 and/or any PilA sequence set forth in SEQ ID NO. 58 — SEQ ID NO. 121) As used herein “signal peptide” refers to a short (less than 60 amino acids, for e, 3 to 60 amino acids) polypeptide present on precursor proteins (typically at the N terminus), and which is typically absent from the mature protein. The signal peptide (sp) is typically rich in hydrophobic amino acids. The signal peptide directs the transport and/or secretion of the translated protein h the membrane. Signal peptides may also be called targeting signals, transit peptides, localization signals, or signal sequences. For example, the signal sequence may be a co-translational or post-translational signal peptide.

A heterologous signal peptide may be cleaved from a fusion protein construct by signal peptide ases during or after protein transportation or ion. For example, the signal peptide peptidase is signal peptide peptidase l. A “heterologous” signal peptide is one which is not ated with the protein as it exists in nature.

As used herein “treatment” means the prevention of occurrence of symptoms of the condition or disease in a subject, the prevention of recurrence of symptoms of the condition or disease in a subject, the delay of recurrence of symptoms of the condition or e in a subject, the decrease in severity or frequency of symptoms of the condition or disease in a subject, slowing or eliminating the progression of the condition and the partial or total elimination of symptoms of the disease or condition in a subject.

As used herein, “optionally” means that the subsequently described s) may or may not occur, and includes both event(s) that occur and events that do not occur.

The pathogenesis of disease caused by NTHi begins with nasopharyngeal colonization.

Mechanisms to adhere to and maintain erm residence within the nasopharyngeal micro- environment are considered ‘virulence determinants’ for NTHi. (Vaccine 28: 279-289 (2010)).

The importance of NTHi being able to adhere to the mucosal epithelial surfaces of a human host is reflected in the multiplicity of adhesins expressed by NTHi. For example, some NTHi express pili. Other adhesive structures belong to the autotransporter family of proteins; these e Hap, HMW1/HMW2 and Hia/Hsf proteins. Further outer membrane proteins, such as the P2 protein, P5 protein and OapA have been described as adhesions for Haemophi/us influenzae. (Cellular Microbiology 4:191-200 , Microbes and Infection 10: 87-96 (2008), e 28: 279-289 ).

Otitis media is a major cause of morbidity in 80% of all children less than 3 years of age.

(Expert Rev. Vaccines 5:517-534 (2006)). More than 90% of children develop otitis media before age 7 (Current Opinion in lnvestigational Drugs 4:953-958 ). In 2000, there were 16 million visits made to -based ians for otitis media in the United States and approximately 13 million antibacterial prescriptions dispensed. (Pediatrics 113:1451-1465 (2004)). In European countries, the reported acute otitis media rates range between 0.125 to 1.24 per year. (Expert Review of es 81479-1500 (2009)). Otitis media is a costly infection and the most common reason children receive antibiotics. (Current ious Disease Reports —182 (2009)). Bacteria are responsible for imately 70% of cases of acute otitis media, with Streptococcus pneumoniae, non-typeable Haemophi/us influenzae, and Moraxe/Ia catarrha/is predominating as the causative agents (Expert Review of Vaccines 5:517-534 (2006)). A subset of children experience recurrent and chronic otitis media and these otitis prone children have protracted middle-ear effusions that are associated with hearing loss and delays in speech and ge development. (Current Infectious e Reports 11:177-182 (2009)). ing the introduction of the heptavalent pneumococcal vaccine in many countries, some studies have demonstrated a significant increase in the proportion of acute otitis media caused by H. zae, with H. influenzae becoming the predominant pathogen. (Pediatric Infectious e Journal -828; Pediatric ious Disease Journal -833 (2004)).

Since otitis media is a multifactorial disease, the feasibility of preventing otitis media using a vaccination strategy has been questioned. (Current Infectious Disease Reports 11:177-182 (2009)). However, the s from one study suggest that it is possible for an n to induce at least partial protection t non-typeable H. influenzae. (Lancet 367:740—748 (2006)). One approach to developing vaccine antigens is to use antigenically conserved regions of genetically heterogeneous but abundantly expressed surface molecules.

Another approach is to identify surface proteins that demonstrate sequence or functional epitope conservation. A third consideration for a vaccine antigen could be to select an antigen that is expressed during infection and colonization in a human host. Murphy (Curr. Infect. e Reports 11:177-182 (2009) states that, despite the existence of several potential non- typeable H. influenzae candidate antigens, one cannot predict with certainty whether the candidate antigen will be ive. (Current Infectious Disease Reports 11:177-182 (2009)).

Some of the proteins bed as potential vaccine antigens are: Haemophi/us adhesin protein (Hap), High molecular-weight (HMVV) proteins 1 and 2, H. influnzae adhesin (Hia), D15 protein, HtrA heat shock protein, P2 surface protein, Iipoprotein D, P5 fimbrin derived peptides, outer membrane protein P4, outer membrane protein (OMP) 26 (OMP26), P6 protein, Protein E, Type IV pilus, Iipooligosaccharide and phosphoryl e. (Current Infectious e Reports 11:177-182 (2009); Expert Review of Vaccines 5:517—534 (2006)).

The chinchilla model is a robust and validated animal model of otitis media and its prevention (Expert Review of Vaccines 8:1063-1082 (2009)). While the illa model may mimic the natural course of human infection, others have suggested that results in the chinchilla model may vary from one laboratory to the next. (Current Opinion in Investigational Drugs 4:953-958 (2003)).

WO 39225 Various other rodents have also been used for the induction of otitis media and are summarized in Vaccine 26:1501-1524 (2008). The murine animal model is often d in otitis media research.

The presence of bactericidal antibody is associated with protection from otitis media due to non-typeable H. influenzae. (Current n in ious e -134 (2003)).

However, an immune response need not be bactericidal to be effective against NTHi.

Antibodies that merely react with NTHi surface adhesins can reduce or eliminate otitis media in the chinchilla. (Current Opinion in lnvestigational Drugs 4:953-958 (2003)).

Chronic obstructive pulmonary disease is a chronic inflammatory disease of the lungs and a major cause of morbidity and mortality worldwide. imately one in 20 deaths in 2005 in the US had COPD as the underlying cause. (Drugs and Aging 26:985-999 (2009)). It is projected that in 2020 COPD will rise to the fifth g cause of disability adjusted life years, chronic invalidating es, and to the third most important cause of mortality (Lancet 349:1498-1504 (1997)).

The course of COPD is characterized by progressive worsening of airflow limitation and a decline in pulmonary function. COPD may be complicated by frequent and recurrent acute exacerbations (AE), which are associated with enormous health care expenditure and high morbidity. (Proceedings of the American Thoracic Society 4:554—564 (2007)). One study suggests that approximately 50% of acute exacerbations of ms in COPD are caused by non-typeable Haemophi/us influenzae, /Ia catarrha/is, Streptococcus pneumoniae, and Pseudomonas nosa. (Drugs and Aging 26:985-999 (2009)). H. influenzae is found in 20-30% of exacerbations of COPD; Streptococcus pneumoniae, in 10-15% of exacerbations of COPD; and Moraxe/la catarrha/is, in 10-15% of exacerbations of COPD.

(New England l of Medicine 359:2355-2365 (2008)). Haemophi/us influenzae, Streptococcus pneumoniae, and Moraxe/Ia catarrha/is have been shown to be the primary pathogens in acute exacerbations of bronchitis in Hong Kong, South Korea, and the Phillipines, while K/ebsie/la spp., Pseudomonas aeruginosa and Acinetobacter spp. constitute a large proportion of pathogens in other Asian countries/regions including lndonesia, Thailand, Malaysia and Taiwan (Respirology, (2011) 16, 532-539; doi:10.1111/j.1440.1843.2011.01943.x). ln Bangladesh, 20% of patients with COPD showed positive sputum e for Pseudomonas, K/ebsie/Ia, Streptococcus niae and Haemophi/us influenzae, while 65% of patients with AECOPD showed positive cultures for Pseudomonas, K/ebsie/Ia, Acinetobacter, Enterobacter, Moraxe/Ia catarrha/is and combinations thereof. (Mymensingh Medical Journal 19:576-585 (2010)). However, it has been ted that the two most important measures to prevent COPD exacerbation are active immunizations and chronic maintenance of cotherapy. (Proceedings of the American Thoracic Society 4:554—564 (2007)).

There is a need for effective vaccines against NTHi. Using antigens that may act at different steps in pathogenesis may improve the efficacy of a vaccine. The inventors have found that PilA and PE may be beneficially present in the immunogenic compositions of the invention as fusion ns.

The present invention relates to fusion proteins of formula (I).

(X) m - (R1)n — A — (Y) o — B — (Z)p (formula I) wherein: X is a signal peptide or MHHHHHH (SEQ ID NO. 2); m is 0 or 1; R1 is an amino acid; n is 0, 1, 2, 3, 4, 5 or6; A is Protein E from Haemophi/us influenzae or an immunogenic nt thereof, or PilA from Haemophi/us zae or an genic fragment thereof; Y is selected from the group consisting of GG, SG, 88 and (G),1 wherein h is 4, 5, 6, 7, 8, 9, or o is 0 or 1; B is PilA from Haemophi/us influenzae or an immunogenic fragment thereof, or Protein E from Haemophi/us influenzae or an immunogenic fragment thereof; Z is HH (SEQ ID NO: 3); and p is 0 or 1.

In one embodiment, the fusion proteins of formula (I) are defined wherein X is selected from the group ting of the signal sequence from CcmH hrome c membrane n H), DsbA (periplasmic protein disulfide isomerise l), DsbB (disulfide bond membrane protein B), FlgI (flagellar peptidoglycan ring protein), FocC (F1c Chaperone protein), MalE (maltose transporter t E), NadA (quinolinate synthase subunit A), NikA (nickel ABC transporter component A), NspA (Neisserial surface protein A), Omp26 (outer membrane protein 26), OmpA (outer ne protein A), OspA (outer surface n A), pelB (pectate lyase B), PhoA (bacterial alkaline phosphatase), PhtD (pneumococcal histidine triad protein D), PhtE ococcal histidine triad protein E), SfmC (periiplasmic pilin chaperone), Sip1 (surface immunogenic protein), TolB (Tol-Pal Cell Envelope x Component B), TorA (trimethylamine e reductase system subunit A), TorT (trimethylamine N-oxide reductase system periplasmic protein T) and Yral (putative periplasmic pilin chaperone); or any subgroup thereof. In one embodiment, X is a co-translational signal peptide or a post-translational signal peptide. In one embodiment X is the signal sequence from FlgI (flgI sp). In another particular ment, X is the signal sequence from pelB (pelB sp). In another embodiment, X is a post-translational signal peptide. In another embodiment, X is selected from the group consisting of the signal sequence from FlgI, NadA and pelB.

In one embodiment, the fusion proteins of formula (I) are defined wherein m is 1. In another embodiment, m is 0.

In one particular embodiment, R1 and n are defined wherein (R1)n is 1 to 6 amino acids enriched in small, usually hydrophilic, amino acids. Hydrophilic amino acids include glutamic acid (E), aspartic acid (D) and asparagine (N).

In one ment, the fusion proteins of formula (I) are defined wherein n is selected from the group consisting of 0, 1, 2 and 6. In one particular embodiment, R1 and n are defined wherein (R1)n is selected from the group consisting of D, E, ATNDDD (SEQ ID NO. 178) and MD, or any subset thereof.

In one particular embodiment, n is ed from the group consisting of 1, 2 and 6. In one particular embodiment, n is 0.

In one embodiment, the fusion ns of formula (I) are defined wherein A is Protein E from H. influenzae. In another ment, the fusion proteins of formula (I) are defined wherein A is Protein E as encoded by an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID WO 39225 NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 5 through SEQ ID NO. 57. In another embodiment, the fusion proteins of formula (I) are defined wherein A is Protein E, wherein Protein E is approximately 75% to 100% identical to the Protein E amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, A is Protein E wherein Protein E is imately 90% to 100% identical to the Protein E amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, A is Protein E wherein Protein E is at least 95% identical to the Protein E amino acid sequence set forth in SEQ ID NO: 4. In additional embodiment, A is Protein E wherein Protein E is at least 95% cal to Protein E as set for in any of SEQ ID NO. 4 — SEQ ID NO. 57. In a ular embodiment, A is Protein E having the amino acid sequence set forth in SEQ ID NO. 4.

In another embodiment, the fusion proteins of formula (I) are d wherein A is an immunogenic fragment of Protein E from H. influenzae. In another embodiment, A is an immunogenic fragment of n E wherein Protein E has an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 4 through SEQ ID NO. 57. In another embodiment, A is an immunogenic fragment of Protein E, wherein Protein E is approximately 75% to 100% identical to the amino acid sequence set forth in SEQ ID NO: 4. In r embodiment, A is an immunogenic fragment of Protein E, n Protein E is approximately 90% to 100% identical to SEQ ID NO. 4. In an additional embodiment, A is an immunogenic fragment of Protein E, wherein Protein E is at least 95% identical to any of SEQ ID NO. 4 — SEQ ID NO. 57. More specifically, in one embodiment, A is an immunogenic fragment of Protein E, n Protein E is 93% to 100% identical to SEQ ID NO. 124. In a particular embodiment, A is an immunogenic fragment of Protein E wherein Protein E is SEQ ID NO. 4.

In another embodiment, A is an immunogenic fragment of Protein E from H. influenzae selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20—160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, A is an immunogenic fragment of Protein E from H. influenzae selected from the group consisting of amino acids 17-160 ofSEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 ofSEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20—160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). In a further embodiment, A is an immunogenic fragment of n E from H. zae selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids -160 ofSEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 ofSEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). More specifically, in one embodiment, A is SEQ ID NO. 124, amino acids 19-160 of SEQ ID NO. 4. In an additional embodiment, A is SEQ ID NO.125, amino acids 20—160 of SEQ ID NO. 5. In another embodiment, A is immunogenic fragment of Protein E from H. influenzae selected from the group consisting of amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180).

Protein E - SEQ ID NO. 4 MK< T.TT.ST. ACSAQ QI A*ZQNDVKL APPTDVRSGY IQLVKNVNYY DSfiS WVD\T QfiL’Q VHE'DA VVNLDKGLYV YPEP<RYARS VQQYKILNCA NY-ILTQVQTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTT. YWAAQ CAN VDK < Amino acids 17-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 122 SAQ QI A*1QNDVKL APPTDVRSGY :RLVKNVNYY DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEPKRYARS VRQYKILNCA NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH DTTL YNAAQ CAN VDK< Amino acids 18-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 123 AQ QIAfiQNDVKL APPTDVRSGY :QLVKNVNYY DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA VQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTL YNAAQ CAN YGEAFSVDK< Amino acids 19-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 124 Q QIAfiQNDVKL APPTDVRSGY :QLVKNVNYY DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTL YNAAQ CAN YGEAFSVDK< Amino acids 20—160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 125 QIAfiQNDVKL APPTDVRSGY :RLVKNVNYY DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS LNCA NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTL YNAAQ CAN YGEAFSVDK< Amino acids 22-160 of Protein E from SEQ ID NO. 4 - SEQ ID NO. 126 KAEQNDVKL APPTDVRSGY :RLVKNVNYY DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA NYiLTQVQTD FYDEFWGQGL RAAP<KQKKH DTTL YNAAQ CAN YGEAFSVDK< Amino acids 23-160 of n E from SEQ ID NO. 4 - SEQ ID NO. 179 AEQNDVKL APPTDVRSGY :RLVKNVNYY DSfiS WVDN QfiBQ VHEDA VVNLDKGLYV YPEP<RYARS VQQYKILNCA 2012/050236 NY-ILTQVQTD FYDEFWGQGL RAAPKKQKKH TLSLTPDTTT. YNAAQ CAN YGEAFSVDK < Amino acids 24-160 Protein E from SEQ ID NO. 4 - SEQ ID NO. 180 EQNDVKL APPTDVRSGY IRLVKNVNYY DSfiS WVDW QfiL’Q VHE'DA VVNLDKGLYV YPEP<RYARS LNCA NY-ILTQVQTD FYDEFWGQGL RAAP<KQKKH TLSLTPDTTT. YWAAQ CAN YGEAFSVDK < In another embodiment, the fusion proteins of formula (I) are defined wherein A is PilA from H. influenzae. In another embodiment, the fusion ns of formula (I) are defined wherein A is PilA from H. influenzae having an amino acid ce selected from the group consisting ofSEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO.111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In another embodiment, A is PilA wherein PilA is approximately 80% to 100% cal to SEQ ID NO. 58. In another ment, A is PilA wherein PilA is at least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121. In a particular embodiment, A is PilA of SEQ ID NO. 58.

In another embodiment, the fusion proteins of formula (I) are defined wherein A an immunogenic fragment of PilA from H. influenzae. In another embodiment, A is an immunogenic fragment of PilA wherein PilA is approximately 80% to 100% identical to SEQ ID NO. 58. For example, A is an immunogenic fragment of PilA wherein PilA has an amino acid ce selected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset SEQ ID NO. 58 through SEQ ID NO. 121. In an additional embodiment, A is an immunogenic nt of PilA wherein PilA is at least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121. In a particular embodiment, A is an immunogenic fragment of PilA from H. influenzae strain 86-028NP n PilA is SEQ ID NO. 58.

PilA from H. influenzae strain 86-028NP - SEQ ID NO. 58 MKLTTQQTL< KGld'TT. 41T.M V A A T.AT A BSYQNYT KKAAVSELLQ ASAPYKADVE LCVYSTWETT NCTGGKNG A AD TTAKGYV KSVTTSWGA: TVKGDGTT.A\T M*'.Y T.QATGN AATGVTWTTT CKGTDASLFP ANFCGSVTQ In another embodiment, A is an genic fragment of PilA approximately 75% to 100% identical to SEQ ID NO. 127. More specifically, in one embodiment A is SEQ ID NO. 127, a fragment consisting of amino acids 40-149 of SEQ ID NO. 58.

Amino acids 40-149 of PilA from H. influenzae strain NP - SEQ ID NO. 127.

T KKAAVSELLQ ASAPYKADVE LCVYSTNETT NCTGGKNG A A3 V KSVTTSWGA: TVKGDGTT.A\T M*'.Y T.QATGN AATGVTWTTT C(GTDASLFP ANFCGSVTQ In another embodiment, A is an immunogenic fragment of PilA consisting of amino acids 40-149 from any of SEQ ID NO. 58 — SEQ ID NO. 121. In an additional embodiment, A is an genic fragment at least 95% identical to amino acids 40-149 from any of SEQ ID NO. 58—SEQ ID NO.121.

In one ment, the fusion proteins of formula (I) are d wherein Y is selected from the group consisting of GG, SG and SS. In another embodiment, the fusion proteins of formula (I) are defined wherein Y is GG or SG. In one particular embodiment, Y is GG.

In one embodiment, the fusion proteins of formula (I) are defined wherein o is 1. In another embodiment, o is 0.

In one embodiment, the fusion proteins of formula (I) are defined wherein B is PilA from H. influenzae or an immunogenic fragment of PilA from H. influenzae when A is n E from H. zae or an immunogenic fragment of Protein E from H. influenzae. For example, B is PilA from H. influenzae strain 86-028NP. In another ment, B is PilA from H. influenzae having an amino acid sequence selected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In another embodiment, B is PilA wherein PilA is approximately 80% to 100% identical to SEQ ID NO. 58. In another embodiment, B is PilA wherein PilA is at least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121. In a particular embodiment, B is PilA ofSEQ ID NO. 58.

In another embodiment, B is PilA wherein PilA is at least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121 and A is PE wherein PE is at least 95% identical to any of SEQ ID NO. 4 — SEQ ID NO. 57.

In another embodiment, the fusion proteins of formula (I) are defined n B is an immunogenic fragment of PilA from H. influenzae when A is an immunogenic fragment of Protein E from H. influenzae. For example, B is an immunogenic fragment of the PilA from H. influenzae strain 86-028N P. In another embodiment, B is an genic fragment of PilA wherein PilA is imately 80% to 100% identical to SEQ ID NO: 58. In another embodiment, B is an immunogenic fragment of PilA wherein PilA has an amino acid selected from the group consisting of SEQ ID NO. 58, SEQ ID NO. 59, SEQ ID NO. 60, SEQ ID NO. 61, SEQ ID NO. 62, SEQ ID NO. 63, SEQ ID NO. 64, SEQ ID NO. 65, SEQ ID NO. 66, SEQ ID NO. 67, SEQ ID NO. 68, SEQ ID NO. 69, SEQ ID NO. 70, SEQ ID NO. 71, SEQ ID NO.72, SEQ ID NO. 73, SEQ ID NO. 74, SEQ ID NO. 75, SEQ ID NO. 76, SEQ ID NO. 77, SEQ ID NO. 78, SEQ ID NO. 79, SEQ ID NO. 80, SEQ ID NO. 81, SEQ ID NO. 82, SEQ ID NO. 83, SEQ ID NO. 84, SEQ ID NO. 85, SEQ ID NO. 86, SEQ ID NO. 87, SEQ ID NO. 88, SEQ ID NO. 89, SEQ ID NO. 90, SEQ ID NO. 91, SEQ ID NO. 92, SEQ ID NO. 93, SEQ ID NO. 94, SEQ ID NO. 95, SEQ ID NO. 96, SEQ ID NO. 97, SEQ ID NO. 98, SEQ ID NO. 99, SEQ ID NO. 100, SEQ ID NO. 101, SEQ ID NO. 102, SEQ ID NO. 103, SEQ ID NO. 104, SEQ ID NO. 105, SEQ ID NO. 106, SEQ ID NO. 107, SEQ ID NO. 108, SEQ ID NO. 109, SEQ ID NO. 110, SEQ ID NO. 111, SEQ ID NO. 112, SEQ ID NO. 113, SEQ ID NO. 114, SEQ ID NO. 115, SEQ ID NO. 116, SEQ ID NO. 117, SEQ ID NO. 118, SEQ ID NO. 119, SEQ ID NO. 120 and SEQ ID NO. 121; or any subset of SEQ ID NO. 58 through SEQ ID NO. 121. In another embodiment, B is an immunogenic nt of PilA wherein PilA is at least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121. In a particular embodiment, B is an immunogenic fragment of PilA from H. influenzae wherein PilA has the amino acid sequence set forth in SEQ ID NO. 58. In another embodiment, B is an immunogenic fragment of PilA consisting of amino acids 40-149 from any of SEQ ID NO. 58 — SEQ ID NO. 121. More specifically, in one embodiment B is the fragment of PilA as set forth in SEQ ID NO. 127. In an additional embodiment, B is an immunogenic fragment at least 95% identical to amino acids 40-149 of any of SEQ ID NO. 58 — SEQ ID NO.121.

In one particular embodiment, B is the fragment of PilA as set forth in SEQ ID NO. 127 and A is an immunogenic fragment of n E selected from the group ting of SEQ ID NO. 122, SEQ ID NO. 124, SEQ ID NO. 125 and SEQ ID NO. 126. More particularly, B is the fragment of PilA as set forth in SEQ ID NO. 127 and A is the fragment of Protein E as set forth in SEQ ID NO. 124, amino acids 19-160 of n E from SEQ ID NO. 4. In another embodiment, B is the fragment of PilA as set forth in SEQ ID NO. 127 and A is the fragment of Protein E as set forth in SEQ ID NO. 125.

In another embodiment, B is an immunogenic fragment of PilA wherein PilA is at least 95% identical to any of SEQ ID NO. 58 — SEQ ID NO. 121 and A is an immunogenic fragment of PE wherein PE is at least 95% identical to any of SEQ ID NO. 4 — SEQ ID NO. 57.

In another embodiment, the fusion proteins of formula (I) are defined wherein B is Protein E from H. influenzae when A is PilA from H. zae. For example, B is Protein E having an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43 SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 4 h SEQ ID NO. 57. In another embodiment, the fusion proteins of formula (I) are defined wherein B is Protein E wherein Protein E is imately 75% to 100% cal to the Protein E amino acid sequence set forth in SEQ ID NO: 4. In another embodiment, B is Protein E wherein Protein E is imately 90% to 100% cal to the Protein E amino acid sequence set forth in SEQ ID NO: 4. For example, B is Protein E wherein Protein E is at least 95% identical to Protein E as set forth in SEQ ID NO. 4. In another embodiment, B is Protein E wherein Protein E is at least 95% identical to any of SEQ ID NO. 4 — SEQ ID NO. 57. In a particular embodiment, B is Protein E having the amino acid sequence set forth in SEQ ID NO. 4.

In another embodiment, the fusion proteins of formula (I) are d wherein B is an immunogenic fragment of Protein E from H. zae when A is an immunogenic fragment of PilA from H. influenzae. For example, B is an immunogenic fragment of Protein E wherein Protein E has an amino acid sequence selected from the group consisting of SEQ ID NO. 4, SEQ ID NO. 5, SEQ ID NO. 6, SEQ ID NO. 7, SEQ ID NO. 8, SEQ ID NO. 9, SEQ ID NO. 10, SEQ ID NO. 11, SEQ ID NO. 12, SEQ ID NO. 13, SEQ ID NO. 14, SEQ ID NO. 15, SEQ ID NO. 16, SEQ ID NO. 17, SEQ ID NO. 18, SEQ ID NO. 19, SEQ ID NO. 20, SEQ ID NO. 21, SEQ ID NO. 22, SEQ ID NO. 23, SEQ ID NO. 24, SEQ ID NO. 25, SEQ ID NO. 26, SEQ ID NO. 27, SEQ ID NO. 28, SEQ ID NO. 29, SEQ ID NO. 30, SEQ ID NO. 31, SEQ ID NO. 32, SEQ ID NO. 33, SEQ ID NO. 34, SEQ ID NO. 35, SEQ ID NO. 36, SEQ ID NO. 37, SEQ ID NO. 38, SEQ ID NO.39, SEQ ID NO. 40, SEQ ID NO. 41, SEQ ID NO. 42, SEQ ID NO. 43, SEQ ID NO. 44, SEQ ID NO. 45, SEQ ID NO. 46, SEQ ID NO. 47, SEQ ID NO. 48, SEQ ID NO. 49, SEQ ID NO. 50, SEQ ID NO. 51, SEQ ID NO. 52, SEQ ID NO. 53, SEQ ID NO. 54, SEQ ID NO. 55, SEQ ID NO. 56 and SEQ ID NO. 57; or any subset of SEQ ID NO. 4 through SEQ ID NO. 57. In another embodiment, the fusion proteins of formula (I) are d n B is an immunogenic fragment of Protein E wherein Protein E is approximately 75% to 100% identical to the Protein E amino acid sequence set forth in SEQ ID NO. 4. In another embodiment, B is an immunogenic fragment of Protein E wherein n E is approximately 90% to 100% identical to the Protein E amino acid sequence set forth in SEQ ID NO: 4. In a particular embodiment, B is an immunogenic fragment of Protein E having the amino acid sequence set forth in SEQ ID NO. 4. In an additional embodiment, B is an immunogenic fragment of Protein E, wherein Protein E is at least 95% cal to any of SEQ ID NO. 4 — SEQ ID NO. 57.

In another embodiment, B is a nt of Protein E from H. influenzae selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125) and amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126). In another embodiment, B is an immunogenic fragment of Protein E from H. zae selected from the group consisting of amino acids 17-160 of SEQ ID NO. 4 (SEQ ID NO. 122), amino acids 18-160 of SEQ ID NO. 4 (SEQ ID NO. 123), amino acids 19-160 of SEQ ID NO. 4 (SEQ ID NO. 124), amino acids 20-160 of SEQ ID NO. 4 (SEQ ID NO. 125), amino acids 22-160 of SEQ ID NO. 4 (SEQ ID NO. 126), amino acids 23-160 of SEQ ID NO. 4 (SEQ ID NO. 179) and amino acids 24-160 of SEQ ID NO. 4 (SEQ ID NO. 180). More specifically, in one embodiment, B is the fragment of Protein E as set forth in SEQ ID NO. 123, amino acids 18-160 of SEQ ID NO. 4.

In one particular embodiment B is an immunogenic fragment of Protein E as set forth in SEQ ID NO. 123, amino acids 18-160 of SEQ ID NO. 4 when A is an immunogenic nt of PilA as set forth in SEQ ID NO. 127.

In one embodiment, the fusion proteins of a (I) are defined wherein p is 0. In another embodiment, the fusion proteins of formula (I) are defined wherein p is 1.

WO 39225 In one embodiment, the fusion protein of formula (I) is selected from the group consisting of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 and SEQ ID NO. 204; or any subset thereof. In another embodiment, the fusion protein of formula (I) is approximately 95% cal to any of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 or SEQ ID NO. 204.

Fusion proteins of formula (I) are useful as immunogens in subjects such as mammals, particularly humans. In particular, the fusion proteins of formula (I) are useful in inducing an immune se against H. influenzae in subjects, particularly humans. More specifically, the fusion proteins of formula (I) are useful in the treatment or prevention of otitis media and/or AECOPD and/or pneumonia.

The present invention relates to immunogenic compositions sing Protein E from H. influenzae (or an immunogenic nt thereof) and PilA from H. influenzae (or an immunogenic fragment thereof), and immunogenic compositions sing fusion proteins of Protein E from H. influenzae (or an immunogenic fragment thereof) and PilA from H. influenzae (or an immunogenic fragment thereof). The present invention also relates to vaccines comprising such genic compositions and therapeutic uses of the same.

In one embodiment, the immunogenic compositions comprise Protein E from H. influenzae (or an immunogenic fragment thereof) and PilA from H. influenzae (or an immunogenic fragment thereof). Protein E may be SEQ ID NO. 4 or a Protein E sequence at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to SEQ ID NO. 4.

The immunogenic nt of Protein E may be SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125 or SEQ ID NO. 126, or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125 or SEQ ID NO. 126. The immunogenic nt of Protein E may be SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 126, SEQ ID NO. 179 or SEQ ID NO. 180 or a sequence having at least 90%, 95%, 96%, 97%, 98%, 99% sequence identity to any one of SEQ ID NO. 122, SEQ ID NO. 123, SEQ ID NO. 124, SEQ ID NO. 125, SEQ ID NO. 126, SEQ ID NO. 179 or SEQ ID NO. 180. Amino acid differences have been described in Protein E from various Haemophi/us species when compared to Protein E from Haemophi/us influenzae Rd as a reference .

Microbes & Infection gendum to “Identification of a novel Haemophi/us influenzae protein important for adhesion to epitheIia cells” [Microbes Infect. 10 (2008) 87-97], available online July 6, 2010, “Article in Press”) provides a sequence for Protein E from H. influenzae strain 772. WO2002/28889 provides a sequence for Protein E from H. zae strain 12085.

Protein E contains an epithelial cell binding region (PKRYARSVRQ YKILNCANYH LTQVR, SEQ ID NO. 128) that has been reported to be conserved among more than 100 clinical NTHi isolates, encapsulated H. influenzae, and e collection strains analyzed (Singh et al, J. Infect. Dis. 201(3):414-9 (2010)). Singh et al. reported that Protein E was highly ved in both NTHi and encapsulated H. zae (96.9% — 100% identity without the signal e). In one embodiment, the fragment of Protein E comprises the binding region of SEQ ID NO. 128 (PKRYARSVRQ YKILNCANYH LTQVR).

PilA is a conserved adhesin expressed in vivo. Full length comparison of 64 sequences of PilA from Haemophi/us influenzae demonstrated approximately 80% to 100% identity.

In another embodiment, the immunogenic composition comprises a fusion protein as defined by formula (I).

In one embodiment, the present immunogenic compositions may be stered with other antigens from H. influenzae. For e, the PE and PilA or the fusion protein of a (I) may be administered with n D from H. zae. Protein D may be as described in WO91/18926. In another embodiment, the immunogenic composition may include the fusion protein of formula (I) and Protein D from H. influenzae.

In another embodiment, the immunogenic compositions of the invention may be administered with additional antigens from other bacterial species also known to cause otitis media, AECOPD or pneumonia.

The amount of the immunogenic composition which is required to achieve the desired therapeutic or biological effect will depend on a number of factors such as the use for which it is ed, the means of administration, the recipient and the type and severity of the condition being d, and will be ultimately at the discretion of the attendant physician or veterinarian. In general, a typical dose for the treatment of a condition caused in whole or in part by H. influenzae in a human, for instance, may be expected to lie in the range of from about 0.003 mg to about 0.090 mg. More specifically, a typical dose for the treatment of a ion caused wholly or in part by H. influenzae in a human may lie in the range of from about 0.01 mg to about 0.03 mg of fusion n. The immunogenic composition may contain additional antigens; a typical dose for the treatment of a condition caused wholly or in part by H. influenzae in a human may lie in the range of from about 0.01 mg to about 0.03 mg for each additional antigen. This dose may be administered as a single unit dose. Several separate unit doses may also be administered. For example, separate unit doses may be administered as separate priming doses within the first year of life or as separate booster doses given at regular intervals (for example, every 1, 5 or 10 .

Formulations comprising the immunogenic compositions of the invention may be d for administration by an appropriate route, for example, by the intramuscular, sublingual, transcutaneous, intradermal or intranasal route. Such formulations may be ed by any method known in the art.

The immunogenic compositions of the t invention may additionally comprise an adjuvant. When the term “adjuvant” is used in this ication, it refers to a substance that is administered in conjunction with the genic composition to boost the patient’s immune se to the immunogenic component of the composition.

Suitable adjuvants e an aluminum salt such as aluminum hydroxide gel or aluminum phosphate or alum, but may also be a salt of calcium, magnesium, iron or zinc, or may be an insoluble suspension of acylated tyrosine, or acylated sugars, cationically or anionically derivatized saccharides, or polyphosphazenes. In one embodiment, the fusion protein, PE or PilA may be adsorbed onto ium phosphate. In another embodiment, the fusion protein, PE or PilA may be adsorbed onto aluminium hydroxide. In a third embodiment, alum may be used as an adjuvant.

Suitable adjuvant systems which promote a predominantly Th1 response include: non- toxic derivatives of lipid A, Monophosphoryl lipid A (MPL) or a derivative thereof, particularly 3- de-O-acylated monophosphoryl lipid A (3D-MPL) (for its preparation see GB 2220211 A); and a combination of monophosphoryl lipid A, preferably 3-de-O-acylated monophosphoryl lipid A, er with either an aluminum salt (for instance aluminum phosphate or aluminum hydroxide) or an oil-in-water on. In such combinations, antigen and 3D-MPL are contained in the same particulate structures, allowing for more efficient delivery of antigenic and immunostimulatory signals. Studies have shown that 3D-MPL is able to further enhance the immunogenicity of an alum-adsorbed antigen (Thoelen et al. Vaccine (1998) -14; EP 689454-81).

SO1 is an Adjuvant System containing MPL (3-O-desacyl-4’- monophosphoryl lipid A), QS21 (Qui/laja saponaria Molina, fraction 21) Antigenics, New York, NY, USA) and liposomes.

A8018 is an Adjuvant System ning MPL, QS21 and liposomes (50 ug MPL and 50 ug QS21). ASO1E is an Adjuvant System containing MPL, QS21 and mes (25 ug MPL and ug QS21). In one embodiment, the immunogenic composition or vaccine ses ASO1.

In another embodiment, the immunogenic composition or vaccine comprises ASO1B or ASO1 E. In a particular embodiment, the immunogenic composition or vaccine comprises ASO1E.

A803 is an Adjuvant System containing d-Tocopherol and squalene in an oil/water (o/w) emulsion. ASO3A is an Adjuvant System containing d-Tocopherol and squalene in an o/w emulsion (11.86 mg tocopherol). A8033 is an Adjuvant System containing pherol and squalene in an o/w emulsion (5.93 mg tocopherol). A8030 is an Adjuvant System containing d- Tocopherol and squalene in an o/w emulsion (2.97 mg tocopherol). In one embodiment, the immunogenic composition or vaccine comprises A803.

A804 is an Adjuvant System containing MPL (50 ug MPL) adsorbed on an aluminum salt (500 ug Al3+ ). In one embodiment, the immunogenic composition or vaccine comprises ASO4.

A system involving the use of QS21 and 3D-MPL is disclosed in WO 94/00153. A composition wherein the QS21 is quenched with cholesterol is disclosed in WO 39. An onal adjuvant formulation involving QS21, 3D-MPL and tocopherol in an oil in water emulsion is described in WO 95/17210. In one embodiment the genic ition additionally comprises a saponin, which may be QS21. The formulation may also comprise an oil in water emulsion and tocopherol (WO 95/17210). Unmethylated CpG containing oligonucleotides (WO 55) and other immunomodulatory oligonucleotides (WO 0226757 and WO 03507822) are also preferential inducers of a TH1 response and are suitable for use in the present invention.

Additional adjuvants are those selected from the group of metal salts, oil in water emulsions, Toll like receptor agonists, (in ular Toll like receptor 2 agonist, Toll like or 3 agonist, Toll like receptor 4 agonist, Toll like receptor 7 agonist, Toll like receptor 8 agonist and Toll like receptor 9 agonist), saponins or combinations thereof.

The present invention provides a s for preparing an immunogenic composition comprising combining a fusion protein of formula (I) with an adjuvant.

The present invention r provides a vaccine ning an immunogenic composition of the invention and a pharmaceutically acceptable excipient.

Possible excipients e arginine, pluronic acid and/or polysorbate. In a preferred embodiment, polysorbate 80 (for example, TWEEN® 80) is used. In a further embodiment, a final concentration of about 0.03% to about 0.06% is used. ically, a final concentration of about 0.03%, 0.04%, 0.05% or 0.06% polysorbate 80 (w/v) may be used.

The present invention provides a process for preparing an genic composition or vaccine comprising combining a fusion protein of formula (I) with a ceutically acceptable excipient.

The present invention also provides nucleic acids encoding the proteins of the invention.

The term “nucleic acid” refers to a polymeric form of nucleotides. tides can be ribonucleotides, deoxyribonucleotides, or modified forms of either ribonucleotides or deoxyribonucleotides. The term includes single and double forms of DNA. The nucleic acids are preferably substantially free from other nucleic acids.

The present invention provides a process of producing nucleic acids of the invention.

Nucleic acids of the invention may be ed by methods known by those skilled in the art.

For example, the c acids of the invention may be synthesized in part or in whole. The nucleic acids may be ed by digesting longer amino acids orjoining shorter amino acids.

The following examples are intended for illustration only and are not intended to limit the scope of the invention in any way.

In the examples, the ing terms have the designated meaning: 6xhis = six histidines; xg = centrifugal force r gravities) ATP = adenosine triphosphate; BCA = bicinchoninic acid; BSA = bovine serum albumin; °C = degrees Celsius; CaClz = calcium chloride; CV = column volume; DNA = deoxyribonucleic acid; DSC = ential scanning calorimetry; DTT = dithiothreitol; dNTP = deoxynucleoside triphosphate; EDTA = ethylenediaminetetraacetic acid; FT = flow through; HCI = hydrogen chloride; His = his = histidine; HEPES = 4-(2-hydroxyethyl)piperazineethanesulfonic acid; IMAC = lized metal ty chromatography; IPTG = isopropyl [3-Dthiogalactopyranoside; KCI = potassium chloride; KZHPO4 = dibasic potassium phosphate; KHZPO4 = monobasic potassium phosphate; LDS = lithium dodecyl sulfate; L = liter; MES = 2—(N-morpholino)ethanesulfonic acid; MgClz = magnesium chloride; ml = iter; RPM = revolutions per minute; min = minute; mM = millimolar; uL = microliter; NaCl = sodium chloride; NazHPO4 = dibasic sodium phosphate; NaHzPO4 = monobasic sodium phosphate; ng = nanogram; nm = nanometer; O/N = overnight; PBS = phosphate ed saline; PCR = polymerase chain reaction; SB = sample buffer; sec = second; w/v = weight/volume.

EXAMPLES Example 1: Fusion proteins Fusion proteins were produced with different signal es and amino acid linker sequences. These fusion proteins allowed for secretion of both Protein E and PilA (or fragments thereof) without being restricted to a single bacterial . The fusion protein is released into the periplasm after removal of the heterologous signal peptide by a signal peptide peptidase. Fusion protein purified from the bacteria does not n the heterologous signal peptide. “Purified” proteins are removed from the bacteria and lack the signal peptide.

The following table describes fusion protein constructs made.

WO 39225 Table 3: Fusion Protein Constructs containing PiIA and n E.

N-terminal -------------------------------------------------------------------------------C-Terminal LVL312 E PilA fragment ProtE fragment GGHHHHHH (A.A.: 40—149 of SEQ ID NO. (AA: 18 to 160 of SEQ ID NO. 4, SEQ 58, SEQ ID NO. 127) ID NO.123) AA. 1 19 21 130 133 275 276 283 LVL291 pelB sp ProtE fragment G PilA fragment GGHHHHHH (AA: 19 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO.

NO. 124 58, SEQ ID NO. 127 A A 1 22 23 164 167 276 277 284 LVL268 pelB sp ProtE fragment G PilA fragment GGHHHHHH (AA: 20 to 160 of SEQ ID NO. 4, SEQ G (A.A.: 40—149 of SEQ ID NO.

ID NO. 125 58, SEQ ID NO. 127 AA. 1 22 24 164 167 276 277 284 LVL269 nadA sp AT ProtE fragment G PilA fragment GGHHHHH ND (AA: 22 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. H DD NO. 12- 58, SEQ ID NO. 127 AA. 1 23 24-29 30 168 171 280 281 288 LVL270 MH ProtE fragment G PilA fragment HH (AA: 17 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 58, HH NO. 122) SEQ ID NO. 127) AA. 1 7 8 151 154 263 LVL315 pelB ProtE fragment GGHHHHHH sp (AA: 22 to 160 of SEQ ID NO. 4, SEQ ID NO. 126) 1 22 25 163 166 275 276 283 LVL317 pelB sp ProtE fragment G PilA fragment (AA: 19 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 124) NO. 58, SEQ ID NO. 127) AA. 1 22 23 164 167 27 LVL318 pelB sp | ProtE fragment G PilA fragment (AA: 22 to 160 of SEQ ID NO. 4, SEQ G (A.A.: 40—149 of SEQ ID ID NO. 126) NO. 58, SEQ ID NO.

AA. 1 22 25 163 166 275 LVL702 pelB sp ProtE fragment GGHHHHHH NO. 125) NO. 58, SEQ ID NO. 127) AA. 1 22 23 163 166 275 283 LVL736 pelB sp ProtE fragment G PilA fragment GG (AA: 17 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 58, HH NO. 122) SEQ ID NO. 127) HH AA. 1 22 23 166 169 278 286 LVL737 pelB Sp ProtE fragment G PilA ragment GGH (AA: 18 to 160 of SEQ ID NO. 4, SEQ ID (A.A.: 40—149 of SEQ ID NO. 58, HHH --I- 22 23 277 285 LVL738 pelB sp ProtE fragment G PilA fragment GGHHHHHH (A.A.: 22 to 160 of SEQ ID NO. 4, G (A.A.: 40—149 of SEQ ID SEQ ID NO. 126) NO. 58, SEQ ID NO. 127) 2223164 LVL739 pelB sp ProtE nt G PilA nt GGHHHHHH (A.A.: 23 to 160 of SEQ ID NO. 4, (A.A.: 40—149 of SEQ SEQ ID NO. 179) ID NO. 58, SEQ ID NO. 127) 22 23 160163 272 LVL740 pelB sp ProtE fragment G PilA fragment GGHHHHHH (A.A.: 24 to 160 of SEQ ID NO. (A.A. 40—149 of SEQ 4, SEQ ID NO. 180) ID NO. 58, SEQ ID NO. 127) 22 23 159162 27 LVL735 pelB sp ProtE fragment G PilA fragment (A.A.: 20 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 125) NO. 58, SEQ ID NO. 127) 22 23 163 LVL778 pelB sp ProtE fragment G PilA fragment (A.A.: 17 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 122) NO. 58, SEQ ID NO. 127) 22 23 LVL779 pelB sp ProtE fragment G PilA fragment (A.A.: 18 to 160 of SEQ ID NO. 4, SEQ ID G (A.A. 40—149 of SEQ ID ) NO. 58, SEQ ID NO.127) 22 23 168 277 LVL780 pelB Sp ProtE fragment G PilA fragment (A.A.: 22 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 126) NO. 58, SEQ ID NO. 127) 22 23 161 164 273 LVL781 pelB sp ProtE fragment G PilA fragment (A.A.: 23 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO.179) NO. 58, SEQ ID NO. 127) 22 23 160 163 272 LVL782 pelB sp ProtE fragment G PilA fragment (A.A.: 24 to 160 of SEQ ID NO. 4, SEQ ID G (A.A.: 40—149 of SEQ ID NO. 180) NO. 58, SEQ ID NO. 127) 22 23 159 1 sp = signal peptide; AA. = amino acid The DNA and amino acid sequences for each of the signal peptides and plasmids listed in Table 3 are set forth below.

SIGNAL SEQUENCES: pe/B signal peptide (DNA) — SEQ ID NO. 129: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcc pe/B signal peptide (Amino Acid) - SEQ ID NO. 130: MKYLLPTAAA GLLLLAAQPA MA FIgI signal peptide (DNA) - SEQ ID NO. 131: atgattaaatttctctctgcattaattcttctactggtcacgacggcggctcaggct FIgI signal peptide (Amino Acid) - SEQ ID NO. 132: M Kb'TSAT. T. T.T.VTTAAQA NadA signal peptide (DNA) - SEQ ID NO. 133: atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcgcactggca NadA signal peptide (Amino Acid) - SEQ ID NO. 134: MKHFPSKVLT TA: LAT FCSG ALA FUSION PROTEIN CONSTRUCT SEQUENCES: The single underlined portion of the amino acid sequences is from PiIA from Haemophi/us influenzae strain NP. The embolded underlined portion of the amino acid sequences was derived from Protein E from Haemophi/us influenza strain 772.

LVL312 (DNA) - SEQ ID NO. 135: aaatttctctctgcattaattcttctactggtcacgacggcggctcaggctgagactaaaaaagcagcggtatctgaattactg caagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatg cagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggat ggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaac ctctttatttccagcaaatttttgcggaagtgtcacacaaggcggcgcgcagattcagaaggctgaacaaaatgatgtgaa gctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtg gataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgca cgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg cgaactatggtgaagcattttcagttgataaaaaaggcggccaccaccaccaccaccactaa LVL312 (protein): (flgI sp)(E)(PiIA aa 40-149)(GG)(ProtE aa 18-160)(GGHHHHHH) - SEQ ID NO. 136 M KETSAL L LLVTTAAQA? TKIAAVS?LL QASAPYKADV ELCVYSTNET KNG RAD TTAKGY VKSVTTSWGA :TVKGDGTLA.WM£Y LQATG NAATGVTWTT TC<GTDASLF PANFCGSVTQ GGAQ QIAfiQ WDVKLAPPTD VRSGYIQLVK NVWYY DSfiS WVDW fiB VNLD <GLYVYPEP< RYARSV? YK :LWCANYiLT VQTDFYDEF WG GLRAAP< < KKHTLSLT PDTTLYWAA AF SVDK<GG€€H HHH LVL291 (DNA) - SEQ ID NO. 137: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccagattcagaaggctgaaca aaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtga atcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcc taaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattt tggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgc tcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtatt gcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggc acattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacgga tgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaa LVL291 (Protein)(pe|B sp)(ProtE aa 19-160)(GG)(PiIA aa40—149)(GGHHHHHH) - SEQ ID NO.

TAAA GLLLLAAQPA NA IAfi N DVKLAPPTDV RSGYIQLVKN VNYY DSfiS WVDW fiB V NLDK GLYVYPEP<R YARSV? YK: LNCANYiLT VQTDFYDEFW G GLRAAP<K KKHTLSLTP DTTLYWAA I "CANYG?AFS VDK<GGT<KA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNG RAD VKSV ITVK GDGTLANMfiY AAT GVTWTTTC<G TDASLFPANF CGSVTQGGHH HiHH LVL268 (DNA) - SEQ ID NO. 139: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgatattcagaaggctgaaca aaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtga atcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcc taaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattt tggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgc tcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtatt gcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggc acattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacgga tgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac LVL268 (protein): (peIB sp)(D)(ProtE aa 20—160)(GG)(PiIA aa40-149)(GGHHHHHH) - SEQ ID NO. 140: MKYLLPTAAA GLLLLAAQPA NAD IAfi N DVKLAPPTDV RSGYIQLVKN VNYY DSfiS WVDW fiB V PEDAVVNLDK GLYVYPEP<R YARSV? YK: LNCANYiLT VQTDFYDEFW G GLRAAP<K KKHTLSLTP AA I "CANYG?AFS VDK<GGT<KA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNG RAD TTA<GYV<SV TTSWGAITVK WMfiY LQATGNAAT GVTWTTTC<G TDASLFPANF CGSVTQGGHH HiHH LVL269 (DNA) - SEQ ID NO. 141: atgaaacactttccatccaaagtactgaccacagccatccttgccactttctgtagcggcgcactggcagccacaaacgacgacg ataaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattatt acatcgatagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtat gtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgat ttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaac gctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtat ctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtg gaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagta gatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttg caaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaa LVL269 (protein): (nadA sp)(ATNDDD)(ProtE aa 22-160)(GG)(PiIA aa 40-149)(GGHHHHHH) - SEQ ID NO.142 MKHFPSKVLT TAILATFCSG DDDK A:L*J ZDVKLAP PTDVRSGYIQ LVKNVNYY D SfiS WVDW fl B VHEDAVV NLDKGLYVYP EP<RYARSVQ YKILNCANY {LT VRTDFY DEFWG GLRA AP<K KKHTL SLTPDTTLYW AA CANYG ?AFSV3<<GG T<KAAVSELL KADV ELCVYSTNET TNCTGGKNG AAD TTA<GY V<SVTTSWGA :TVKGDGTLA.WM£Y LQATG NAATGVTWTT TC<GTDASLF PANFCGSVTQ GGHHHiHH LVL270 (DNA) - SEQ ID NO. 143: atgcaccaccaccaccaccacagcgcgcagattcagaaggctgaacaaaatgatgtgaagctggcaccgccgactgatgtacg aagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggataaccaagagccacaaattgta cattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgttctgttcgtcagtataagatcttg aattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgcgggcagcacctaaaaagca aaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcgaactatggtgaagcattttcagtt gataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttataaggctgatgtggaattatgtgt atatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataaccacagcaaaaggctatgtaa aatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatggaatatattttgcaagctacag gtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccagcaaatttttgcggaagtgtcac acaataa LVL270 (protein): (MHHHHHH)(ProtE aa 17-160)(GG)(PiIA aa40-149) - SEQ ID NO. 144.- WHiiiHHSA IAfi NDVK LAPPTDVQSG YIQLVKNVNY Y DSfiS WVD W fiB VHED AVVNLDKGLY VYPEP<RYAR SV? YKILNC ANYiLT VAT DFYDEFWG G LRAAP<K KK HTLSLTPDTT LYWAA CA NYGEAFSVDK jGGT<KAAVS ELLQASAPYK VYST TGGK NG AAD TTA <GYV<SVTTS WGAITVKGDG TLAWMfiY LQ ATGNAATGVT GTDA SLFPANFCGS VTQ LVL315 (DNA) - SEQ ID NO. 145: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaa tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa cgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt cagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca gcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacat tggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcc tctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccactaa LVL315 (protein): (peIB sp)(M D)(ProtE aa 22-160)(GG)(PiIA aa40—149)(GGHHHHHH) - SEQ ID NO. 146: MKYLLPTAAA GLLLLAAQPA MAMDKAE ND VKLAPPTDV? SGYIQLVKNV NYY DSfiS W VD\T *ZL’ VH b'DAVVNLDKG P<RY ARSV? YKIL NCANY-ILT V QTDFYDEFWG GLQAAP<K KKHTLSLTPD TTT.Y\TAA AFSV DK<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG GKNG AAD T TA<GYV<SVT TVKG DGTT.A\IM*ZY T.QATGNAATG VTWTTTC<GT DASLFPANFC GSVTQGGHHH -IHH LVL317 (DNA) - SEQ ID NO. 147: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggcccagattcagaaggctgaaca tgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtga atcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcc taaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattt tggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgc tcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaa gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtatt gcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggc acattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacgga tgcctctttatttccagcaaatttttgcggaagtgtcacacaataa LVL317 (protein): (peIB sp)(ProtE aa 19-160)(GG)(PiIA aa40-149) - SEQ ID NO. 148: MKYLLPTAAA GLLLLAAQPA NA IAfi N DVKLAPPTDV RSGYIQLVKN VNYY DSfiS WVDW fiB V PEDAVVNLDK GLYVYPEP<R YARSV? YK: iLT VQTDFYDEFW G GLRAAP<K KKHTLSLTP DTTLYWAA I "CANYG?AFS VDK<GGT<KA AVSELLQASA ELCV YSTNETTNCT GGKNG RAD TTA<GYV<SV TTSWGAITVK GDGTLAWMfiY LQATGNAAT GVTWTTTC<G TDASLFPANF CGSVTQ LVL318 (DNA) - SEQ ID NO. 149: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccatggataaggctgaacaaaa tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa cgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt cttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca gcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacat tggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcc tctttatttccagcaaatttttgcggaagtgtcacacaataa LVL318 in): (peIB sp)(M D)(ProtE aa 22-160)(GG)(PiIA aa40-149) - SEQ ID NO. 150.- MKYLLPTAAA GLLLLAAQPA MAMDKAL‘LJ ND VKLAPPTDVR VKNV NYY DSfiS W VDW fiB VH EDAVVNLDKG LYVYPEP<RY ARSV? YKIL NCANYiLT V QTDFYDEFWG GLRAAP<K KKHTLSLTPD TTLYWAA CANYGEAFSV DK<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG GKNG AAD T TA<GYV<SVT TSWGAITVKG DGTLAWMfiY AATG VTWTTTC<GT DASLFPANFC GSVTQ LVL702 (DNA) - SEQ ID NO. 181: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccattcagaaggctgaacaaaa tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa gcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt cagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca gcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacat tggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcc tctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac LVL702 (protein): (peIB sp)(ProtE aa 20-160)(GG)(PiIA aa40-149)(GGHHHHHH) - SEQ ID NO. 182: MKYLLPTAAA GLLLLAAQL’A MA IAfi ND VKLAPPTDV? SGYIQLVKNV NYY DSfiS W VD\T *ZL’ VH b'DAVVNLDKG LYVYPEP<RY ARSV? YKIL NCANY-ILT V QTDFYDEFWG GLRAAP<K KKHTLSLTPD TAA CANYGEAFSV DK<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG GKNG AAD T TA<GYV<SVT TSWGAITVKG DGTT.A\IM*ZY T.QATGNAATG VTWTTTC<GT DASLFPANFC GSVTQGGHHH -IHH LVL736 (DNA) - SEQ ID NO. 183: tacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggc tgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcga tagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcct gagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgat gaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataa tgctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaatta gcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaa atggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggg gatggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaagg aacggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac LVL736 (protein): (peIB sp)(ProtE aa 17-160)(GG)(PiIA aa40—149)(GGHHHHHH) - SEQ ID NO. 184: TAAA GLLLLAAQPA MASAQ QIAfi QNDVKLAPPT DVRSGYIQLV KNVNYY DSfi S WVDW fiB VHEDAVVNL DKGLYVYPEP {RYARSVQ Y KILNCANYiL T VQTDFY37L‘L FWG GLRAAP {K {KHTLSL TPDTTLYWAA CANYG?A GGT< KAAVSELLQA SAPYKADVEL CVYSTNETTN CTGGKNG AA 3 TTA<GYV< SVTTSWGAIT LAWM fiY LQATGNA ATGVTWTTTC {GTDASLFPA NFCGSVTQGG HHHiHH LVL737 (DNA) - SEQ ID NO. 185: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctga tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatag tgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctga gcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatga attttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgc tgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactg caagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatg gtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggat ggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaac ggatgcctctttatttccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac LVL737 (protein): (pe/B sp)(ProtE aa 18-160)(GG)(Pi/A aa40—149)(GGHHHHHH) - SEQ ID NO. 186: MKYLLPTAAA GLLLLAAQPA MAAQ QIAfiQ NDVKLAPPTD QLVK NVNYY DSfiS WVDW fiB VHEDAVVNLD KGLYVYPEP< RYARSV? YK :LNCANYiLT VQTDFYDEF WG GLRAAP< K {KHTLSLT WAA CANYG?AF SVDK<GGT<K AAVSELLQAS APYKADVELC VYSTNETTNC TGGKNG AAD TTA<GYV<S AITV KGDGTLAWMfi Y LQATGNAA TGVTWTTTC< GTDASLFPAN FCGSVTQGGH HHiHH WO 39225 LVL738 (DNA) - SEQ ID NO. 187: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgt gaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctg ggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgtta tgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggaca gggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatt tgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagc gccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcag atataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggca aatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatt tccagcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac LVL738 (protein): (pe/B sp)(ProtE aa 22-160)(GG)(Pi/A aa40—149)(GGHHHHHH) - SEQ ID NO. 188: MKYLLPTAAA GLLLLAAQPA MAKAL*J ZDVK LAPPTDVRSG YIRLVKNVNY Y DSfiS WVD W fiB VHED AVVNLDKGLY VYPEP<RYAR SV? YKILNC ANYiLT VQT DFYDEFWG G LRAAP<K KK HTLSLTPDTT LYWAA CA NYGEAFSVDK <GGT<KAAVS ELLQASAPYK ADVELCVYST NETTNCTGGK NG AAD TTA VTTS KGDG TLAWMfiY LQ ATGNAATGVT GTDA SLFPANFCGS VTQGGHHHiH H LVL739 (DNA) - SEQ ID NO. 189: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgctgaacaaaatgatgtgaa gctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtg gataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgca cgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt ttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg cgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgcct tataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatat aaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaat atggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttcc agcaaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac LVL739 (protein): (pe/B sp)(ProtE aa 23-160)(GG)(Pi/A 49)(GGHHHHHH) - SEQ ID NO. 190.- M<YLLPTAAA GLLLLAAQPA MARL*J ZDVKL APPTDVRSGY :QLVKNVNYY DSfiS WVDW fiB VHEDA VVNLDKGLYV YPEP<RYARS V? YKILNCA NYiLT VQTD FYDEFWG GL RAAP<K {KH TLSLTPDTTL YWAA CAN YGEAFSVDK< GGT<KAAVSE.L LLQASAPYKA DVELCVYSTN ETTNCTGGKN G RAD TTA< GYV<SVTTSW GAITVKGDGT LAWMfiY LQA TGNAATGVTW TDAS LFPANFCGSV TQGGHHHiHH LVL740 (DNA) - SEQ ID NO. 191: tacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagct ggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggat aaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgt tctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttg cgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcg aactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttat aaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataac cacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatg gaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccag caaatttttgcggaagtgtcacacaaggcggccaccaccaccaccaccac LVL740 (protein): (pe/B sp)(ProtE aa 24-160)(GG)(Pi/A aa40—149)(GGHHHHHH) - SEQ ID NO. 192.- M<YLLPTAAA GLLLLAAQPA MAIL*J ZDVKLA PPTDVQSGY: QLVKNVNYY: iSfiS WVDWD fiB VHEDAV VNLDKGLYVY PEP<RYARSV Q YKILNCAN YiLT VQTDF YDEFWG GLR AAP<K KKHT LSLTPDTTLY WAA CANY GEAFSVDK<G GT<KAAVSEL LQASAPYKAD STNE TTNCTGGKNG RAD TTA<G YV<SVTTSWG AITVKGDGTL AWMfiY LQAT VTWT TTC<GTDASL GSVT QGGHHHiHH LVL735 (DNA) - SEQ ID NO. 193: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccattcagaaggctgaacaaaa tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcg atctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaa cgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggg gacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcag attatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgt cagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgca ataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacatt ggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcct ctttatttccagcaaatttttgcggaagtgtcacacaa LVL735 (protein): (pe/B otE aa 20-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 194.- MKYLLPTAAA GLLLLAAQL’A MA 521 A*1§2ND VKLAPPTDVR VKNV NYY DSfiS W VD\T *ZL’ VH b'DAVVNLDKG LYVYPEP<RY ARSV? YKIL NCANY-ILT V QTDFYDEFWG GLRAAP<K KKHTLSLTPD TTT.Y\TAA AFSV D<<GGT<KAA VSELLQASAP YKADVELCVY STNETTNCTG GKNG AAD T TA<GYV<SVT TSWGAITVKG DGTT.A\IM*ZY T.QATGNAATG VTWTTTC {GT DASLFPANFC GSVTQ LVL778 (DNA) - SEQ ID NO. 195: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccagcgcccagattcagaaggc tgaacaaaatgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcga tagtgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcct gagcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgat gaattttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataat gctgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattac tgcaagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaat ggtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaagggg atggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaagga acggatgcctctttatttccagcaaatttttgcggaagtgtcacacaa VV()2012/139225 LVL778 (protein): (pe/B sp)(ProtE aa )(GG)(Pi/A aa40—149) - SEQ ID NO. 196: MKYLLPTAAA GLLLLAAQPA NASA IA*'. NDVKLAPPT DVQSGYIQLV KNVNYY DSfi S WVDW fiB VNL DKGLYVYR {RYARSVQ Y KILNCANYiL T VQTDFY37L‘L FWG GLRAAP {K KKHTLSL TPDTTLYWAA CANYG?A FSV3K<GGT< KAAVSELLQA SAPYKADVEL CVYSTNETTN G AA 3 TTA<GYV< SVTTSWGAIT VKG:DGTLAWM fiY A ATGVTWTTTC {GTDASLFPA NFCGSVTQ LVL779 (DNA) - SEQ ID NO. 197: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgcccagattcagaaggctga tgatgtgaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatag tgaatcgatctgggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctga gcctaaacgttatgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatga attttggggacagggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgc tgctcagattatttgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactg caagcgtcagcgccttataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatg gtattgcagcagatataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggat ggcacattggcaaatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaac ggatgcctctttatttccagcaaatttttgcggaagtgtcacacaa LVL779 (protein): (pe/B sp)(ProtE aa 18-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 198: MKYLLPTAAA GLLLLAAQPA MAA I Afi NDVKLAPPTD VQSGYIQLVK NVNYY DSfiS WVDW fiB VHEDAVVNLD KGLYVYPEP 4 ? YK :LNCANYiLT VQTDFYDEF WG GLRAAP < K KKHTLSLT PDTTLYWAA CANYG?AF SVDK<GGT<K AAVSELLQAS APYKADVELC VYSTNETTNC TGGKNG AAD TTA<GYV<S VTTSWGAI:TV KGDGTLAWMZ4 Y LQATGNAA TGVTWTTTC< GTDASLFPAN FCGSVTQ LVL780 (DNA) - SEQ ID NO. 199.‘ atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccaaggctgaacaaaatgatgt gaagctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctg ggtggataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgtta tgcacgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggaca gggtttgcgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatt tgtgcgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagc taaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcag atataaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggca aatatggaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatt tccagcaaatttttgcggaagtgtcacacaa LVL780 (protein): (pe/B sp)(ProtE aa 22-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 200.- MKYLLPTAAA GLLLLAAQPA MAKAES 2NDVK LAPPTDVQSG YIQLVKNVNY Y DSfiS WVD \T *1? VHE'D AVVNLDKGLY VYPEPKRYAR SV? YKILNC ANY-1LT VQT DFYDEFWG G LRAAP<K KK HTLSLTPDTT TIYWAA CA NYGEAFSVDK <GGT<KAAVS ELLQASAPYK ADVELCVYST NETTNCTGGK NG RAD TTA <GYV<SVTTS \TGAITVKGDG TT.A\]M*'.Y T.Q ATGNAATGVT WTTTC<GTDA SLFPANFCGS VTQ LVL781 (DNA) - SEQ ID NO. 201: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgctgaacaaaatgatgtgaa gctggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtg gataaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgca cgttctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggt gcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtg cgaactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgcct tataaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatat aaccacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaat tatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttcc agcaaatttttgcggaagtgtcacacaa LVL781 (protein): (pe/B sp)(ProtE aa 23-160)(GG)(Pi/A aa40—149) - SEQ ID NO. 202.- M<YLLPTAAA AQPA MAAE NDVKL APPTDVRSGY :QLVKNVNYY DSfiS WVDW fiB VHEDA VVNLDKGLYV YPEP<RYARS V? YKILNCA NYiLT VQTD FYDEFWG GL RAAP<K KKH TLSLTPDTTL YWAA CAN YGEAFSV3<< AVSE.L LLQASAPYKA DVELCVYSTN ETTNCTGGKN G AAD TTA< TTSW GAITVKGDGT LAWMfiY LQA TGNAATGVTW TTTC<GTDAS LFPANFCGSV TQ LVL782 (DNA) - SEQ ID NO. 203: atgaaatacctgctgccgaccgctgctgctggtctgctgctcctcgctgcccagccggcgatggccgaacaaaatgatgtgaagct ggcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggat aaccaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgt tctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttg cgggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcg aactatggtgaagcattttcagttgataaaaaaggcggcactaaaaaagcagcggtatctgaattactgcaagcgtcagcgccttat aaggctgatgtggaattatgtgtatatagcacaaatgaaacaacaaactgtacgggtggaaaaaatggtattgcagcagatataac cacagcaaaaggctatgtaaaatcagtgacaacaagcaacggtgcaataacagtaaaaggggatggcacattggcaaatatg gaatatattttgcaagctacaggtaatgctgcaacaggtgtaacttggacaacaacttgcaaaggaacggatgcctctttatttccag caaatttttgcggaagtgtcacacaa LVL782 (protein): (pe/B sp)(ProtE aa 24-160)(GG)(Pi/A 49) - SEQ ID NO. 204.- TAAA GLLLLAAQPA MAIL‘LJ NDVKLA PPTDVRSGY: QLVKNVNYY: DSfiS WVDW fiB VHEDAV VNLDKGLYVY PEP<RYARSV Q YKILNCAN YiLT VQTDF YDEFWG GLR AAP<K {KHT LSLTPDTTLY WAA CANY GEAFSVDK<G GT<KAAVSEL LQASAPYKAD VELCVYSTNE TTNCTGGKNG AAD TTA<G YV<SVTTSWG AITVKGDGTL AWMfiY LQAT GNAATGVTWT TTC<GTDASL FPANFCGSVT Q WO 39225 The full length sequence for PE and PilA from which the above sequences were obtained are set forth in SEQ ID NO. 4 (PE) and SEQ ID NO. 58 (PilA), respectively.

Example 2: Vector Construction and Transformation Primers for amplifying PE from H. influenzae strain 772 were designed based on the sequence of H. influenzae strain Hi Rd. The 5’ primer sequence contains one nucleotide difference compared to the NTHi 772 sequence, introducing an amino acid difference at position 24 when compared with the currently reported NTHi 772 genome sequence. Amino acid #24 in the fusion protein constructs is E (glutamic acid) instead of K (lysine) as found in NTHi 772.

DNA Sequence for PE from H. influenzae strain Rd. - SEQ ID NO. 151 atgaaaaaaattattttaacattatcacttgggttacttaccgcttgttctgctcaaatccaaaaggctgaacaaaatgatgtgaagctg gcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggata agccacaaattgtacattttgatgctgtggtgaatttagataggggattgtatgtttatcctgagcctaaacgttatgcacgttc tgttcgtcagtataagattttgaattgtgcaaattatcatttaactcaaatacgaactgatttctatgatgaattttggggacagggtttgcg ggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcaaat aaagcattttcagttgataaaaaataa n Sequence for PE from H. influenzae strain Rd. - SEQ ID NO. 152 M<K T.TT.ST. GT.T.TACSAQ QI AfiQNDVKL APPTDVRSGY IRLVKNVNYY DSfiS WVDW QfiL’Q VHE'DA VVNLDRGLYV YPEP<RYARS VQQYKILNCA NYHLTQHQTD li'YDL'lr'WGQGL RAAP<KQKKH TLSLTPDTTT. YWAAQ CAN YGKAFSVDK 4 DNA Sequence for PE from H. influenzae strain 772 (as set forth in: Microbes & ion, Corrigendum to ”Identification of a novel Haemophi/us influenzae protein important for adhesion to epithe/ia cells” [Microbes Infect. 10 (2008) 87-97], available online July 6, 2010, ”Article in ) - SEQ ID NO. 153 atgaaaaaaattattttaacattatcacttgggttacttactgcctgttctgctcaaatccaaaaggctaaacaaaatgatgtgaagctg gcaccgccgactgatgtacgaagcggatatatacgtttggtaaagaatgtgaattattacatcgatagtgaatcgatctgggtggata accaagagccacaaattgtacattttgatgcagtggtgaatttagataagggattgtatgtttatcctgagcctaaacgttatgcacgtt ctgttcgtcagtataagatcttgaattgtgcaaattatcatttaactcaagtacgaactgatttctatgatgaattttggggacagggtttgc gggcagcacctaaaaagcaaaagaaacatacgttaagtttaacacctgatacaacgctttataatgctgctcagattatttgtgcga gtgaagcattttcagttgataaaaaa Protein Sequence for PE from H. influenzae strain 772 (as set forth in: Microbes & ion, Corrigendum to ification of a novel Haemophilus influenzae protein important for adhesion to epithe/ia cells” [Microbes Infect. 10 (2008) 87-97], available online July 6, 2010, ”Article in Press’)) - SEQ ID NO. 154 M<< T.TT.ST. GT.T.TACSAQI QKAKQNDVKL APPTDV’RSGY IRLVKNVNYY DSfiS WVDW QfiL’Q VHE'DA VVNLDKGLYV YPEPKRYARS VQQYKILNCA NY-ILTQVQTD GQGL RAAP<KQKKH TLSLTPDTTT. YWAAQ CAN VDK < Vector construction: To generate LVL312, LVL291, LVL268, LVL269, LVL270, LVL702, , , LVL779, LVL780, LVL781 and LVL782, a polymerase chain reaction (PCR) preparation of the following components was prepared (specific components are subsequently exemplified): 36.6 pl of deionized water, 5 pl of buffer #1 10X, 5 pl of dNTPs 2mM, 2 pl MgClz 25 mM, 0.4 pl of primer #1 (50 pM), 0.4 pl of primer #2 (50 pM), 0.5 pl of template (100 ng/pl) and 0.4 pl of KOD HiFi DNA polymerase 2.5 units/pl (NOVAGEN®) was formulated. Polymerase chain reaction involved cycles of 15 seconds of denaturation at 98°C, 2 seconds for annealing at 55°C and 20 seconds of primer extension at 72°C. The PCR products were purified using QIAQUICK® PCR purification kit (QIAGEN®). This product was used under conditions recommended by the supplier which were: the addition of 5 volumes Buffer PB, provided in the QIAQUICK® PCR purification kit, to 1 volume of the PCR preparation. The PCR preparation with Buffer PB was subsequently mixed by vortex. A CK® column was placed into a 2 ml collection tube. To bind DNA in the PCR preparation to the column, the mixed sample was applied to the QIAQUICK ® column and centrifuged for 30—60 seconds at 14 000 RPM. The hrough was discarded and the QIAQUICK ® column was placed back in the same tube. To wash the bound DNA 0.75 ml Buffer PE, provided in the QIAQUICK ® PCR cation kit, was added to the CK ® column, and the column was centrifuged for 30—60 seconds at 14 000 RPM. The flow-through was discarded and the QIAQUICK ® column was placed back in the same tube.

The QIAQUICK ® column was centrifuged once more in the 2 ml collection tube for 1 minute to remove residual wash buffer. Each QIAQUICK ® column was placed in a clean 1.5 ml microcentrifuge tube. To elute the DNA, 33 pl water was added to the center of the QIAQUICK ® membrane and the column was centrifuged for 1 minute at 14 000 RPM. Restriction enzymes and buffer related were obtained from New England BioLabs. For example, approximately 5 pl of pET26b vector (100 ng/pl), 2 pl of NEBuffer 2 (New England Biolabs, 1X NEBuffer 2: 50 mM NaCl, 10 mM Tris-HCl, 10 mM MgCl2, 1 mM dithiothreitol, pH 7.9 at 25°C), 1 pl of Nde/ (20 000 units/ml), 1 pl of Hind/ll (20 000 units/ml) and 11 pl of deionized water were mixed and incubated for two hours at 37°C for DNA digestion. Thereafter, a second step of purification was performed using the QIAQUICK ® PCR purification kit (QIAGEN®) with the procedure described above.

Ligation was performed using Quick T4 DNA ligase and Quick Ligation on Buffer from New England s. For example, around 10 ng of vector and 30 ng of insert in 10 pl of deionized water were mixed with 10 pl of 2X Quick Ligation Reaction Buffer (New England Biolabs, 132 mM Tris-HCl, 20 mM MgClz, 2mM dithiothreitol, 2 mM ATP, 15% polyethylene glycol, pH 7.6 at 25°C) and 1 pl of Quick T4 DNA ligase (New England s). The enzymatic reaction was incubated for 5 s at room temperature before ormation.

To generate , LVL317, LVL318, LVL736, , LVL738, LVL739 and LVL740, a PCR preparation of the following components was prepared: 40 pl of deionized water, 5 pl of reaction buffer 10X, 1 pl of dNTPs mix, 1 pl of primer #1 (10 pM), 1 pl of primer #2 (10 pM), 1 pl of te (25 ng/pl) and 1 pl of Pqu/tra High-Fidelity DNA polymerase 2.5 units/pl (QuikChange ll Site-Directed Mutagenesis Kit, t Technologies, Stratagene Division) was formulated.

Polymerase chain on involved one cycle of denaturation at 95°C for 30 sec, 18 cycles of sec of denaturation at 95°C, 1 min for annealing at 55°C and 5 min 30 sec of primer extension at 68°C. The PCR products were digested using 1 pl of Dpnl restriction enzyme at 37°C for one hour before transformation.

A detailed list of PCR primer sequences used for amplifications is illustrated in Table 4.

To generate pRlT16711, the PE gene fragment coding for amino acids 22 to 160 of SEQ ID NO. 4, which excludes the sequence coding for its corresponding secretion signal, was amplified by PCR from genomic DNA of NTHi strain 772. The amplification primers were designed based on the available strain Hi Rd sequence (at that time, the 772 sequence was not . The 5’ primer sequence contains one mutation compared to the NTHi 772 sequence nce as now available), introducing one amino acid difference in PE coding sequence at position 24, glutamic acid (E) instead of lysine (K). After PCR amplification, the insert was cloned in the pET-26(+) expression vector (NOVAGEN®) using BamHI and Xhol restriction sites.

To generate pRlT16671, a DNA nt coding for a PilA gene fragment (amino acids 40 to 149 of SEQ ID NO. 58, SEQ ID NO. 127), which excludes its leader peptide as well as a portion of the predicted hobic alpha helix, was amplified from genomic DNA of NTHi strain 86- 028NP and cloned into the pET15 expression vector. The vector pRlT16790 (containing amino acids 40 to 149 from NTHi strain 86-028NP) was used as a template to generate the vector 671. The PilA gene fragment was amplified by PCR using the vector pRlT16790 and primers MDES PlLA-3 and MDES . The Pi/A fragment was cloned into the pET-26 expression vector using Nde/ / Xhol ction sites. The DNA sequence encoding six histidine (his) amino acids was incorporated into the 5’ primer to add six histidines (6xhis) at the N- terminal end of the PilA sequence (MDES PlLA-3).

To generate LVL312 (Flgl signal peptide-E-PilA fragment-GG-PE fragment-GGHHHHHH), a polymerase chain reaction was performed to amplify the PilA gene (amino acids 40-149 / strain 86-028NP) using the pRlT16671 vector as a template and s CAN534 and CAN537. DNA sequence corresponding to Fig] signal peptide (sp) and glutamic acid (E) amino acid was incorporated into the 5’ primer (CAN534). To link the PilA ce to PE sequence, DNA sequence corresponding to the two glycine (GG) amino acids linker and the N-terminal PE amino acids were incorporated into the 3’ primer 7). Another polymerase chain reaction was performed to amplify the PE gene (amino acids 18-160) using pRlT16711 vector as a te and primers CAN536 and CAN538. DNA ce corresponding to the C-terminal PilA amino acids and GG amino acids were incorporated into the 5’ primer to link pilA to PE sequence (CAN536). DNA sequence corresponding to the GG amino acids linker and 6xhis amino acids were incorporated into the 3’ primer (CAN538). Finally, to generate LVL312, a third polymerase chain reaction was performed to amplify the PilA and PE genes in fusion with the Fig] signal peptide at the N-terminus, a glutamic acid (E) amino acid between Fig] and pilA, a GG linker between PilA and PE sequences and a GG linker between PE and the 6xhis amino acids at the C-terminus. To e this amplification, the products of the two polymerase chain reactions described above were used as a template with primers CAN534 and CAN538. DNA sequence corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll restriction site was incorporated into the 3’ . The generated PCR product was then inserted into the pET-26b(+) cloning vector (NOVAGEN®).

To generate LVL291 (pelB signal peptide-PE fragment-GG-PilA fragment-GG-6xhis), a polymerase chain reaction was performed to amplify the PE gene (amino acids ) using the pRlT16711 vector as a template and primers CAN544 and CAN546. DNA sequence corresponding to pelB signal peptide (sp) amino acids was incorporated into the 5’ primer (CAN544). To link the PilA sequence to the PE sequence, DNA sequence ponding to GG amino acids linker and the inal PilA amino acids were incorporated into the 3’ primer (CAN546). Another polymerase chain reaction was med to amplify the PilA gene (amino acids 40-149 of SEQ ID NO. 58, SEQ ID NO. 127) using the pRlT16671 vector as a template with primers CAN545 and CAN535. DNA sequence corresponding to the C-terminal PE amino acids and GG amino acids were incorporated into the 5’ primer (CAN545) to link the PilA sequence to the PE sequence. DNA ce corresponding to linker GG amino acids and 6xhis amino acids were incorporated into the 3’ primer (CAN535). Finally, to generate LVL291, a third polymerase chain reaction was performed to amplify the PE and PilA genes in fusion with the pelB signal peptide at the N-terminus, a GG linker between the PE and PilA sequences and a GG linker between PilA and 6xhis amino acids at the C-terminus. To achieve this amplification, the products of two polymerase chain reactions described above were used as a template with primers CAN544 and CAN535. DNA sequence corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll restriction site was incorporated into the 3’ primer. The ted PCR product was then inserted into the pET-26b(+) cloning vector (NOVAGEN®).

To generate LVL268 (pelB signal peptide-D-PE fragment-GG-PilA fragment-GG-6xhis), a rase chain reaction was performed to amplify the PE gene (amino acids 20-160) using the pRlT16711 vector as a template with primers CAN547 and CAN546. DNA sequence corresponding to the pelB signal peptide (sp) amino acids and aspartic acid (D) amino acid were incorporated into the 5’ primer (CAN547). To link the PilA ce to the PE sequence, DNA sequence corresponding to GG amino acids linker and the N-terminal PilA amino acids were incorporated into the 3’ primer (CAN546). Another polymerase chain reaction was performed to y the PilA gene (amino acids 40-149 / NTHi strain 86-028NP) using the pRlT16671 vector as a template with CAN545 and CAN535. DNA ce corresponding to the C-terminal PE amino acids and GG amino acids were orated into the 5’ primer (CAN545) to link the PilA sequence to the PE sequence. DNA sequence corresponding to linker GG amino acids and 6xhis amino acids were incorporated into the 3’ primer (CAN535). Finally, to generate LVL268, a third polymerase chain reaction was med to amplify the PE and PilA genes in fusion with the pelB signal peptide at the N-terminus, a D amino acid between pelB signal e and PE, a GG linker between PE and pilA sequences and a GG linker between PilA and 6xhis amino acids in C-term. To achieve this amplification, the products of the two polymerase chain reactions described above were used as a template with primers CAN547 and CAN535. DNA sequence corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll restriction site was incorporated into the 3’ primer. The generated PCR product was then ed into the pET-26b(+) cloning vector (NOVAGEN®).

To generate LVL269 (NadA signal peptide-ATNDDD-PE nt-GG-PilA fragment-GG-6xhis), a polymerase chain reaction was performed to amplify the PE gene (amino acids 22-160 of SEQ ID NO. 4) using the pRlT16711 vector as a template with primers CAN548 and CAN546.

DNA sequence corresponding to pelB signal e (sp) amino acids and ATNDDD amino acids were incorporated into the 5’ primer (CAN548). To link the PilA sequence to the PE sequence, DNA ce corresponding to the GG amino acids linker and the inal PilA amino acids were incorporated into the 3’ primer (CAN546). Another polymerase chain reaction was performed to amplify the Pi/A gene (amino acids 40-149 of SEQ ID NO. 58, SEQ ID NO. 127) using the pRlT16671 vector as a te with primers CAN545 and CAN535. DNA sequence corresponding to the C-terminal PE amino acids and GG amino acids were orated into the 5’ primer to link the PilA sequence to the PE sequence (CAN545). DNA sequence ponding to linker GG amino acids and 6xhis amino acids were incorporated into the 3’ primer (CAN535). Finally, to generate LVL269, a third polymerase chain reaction was performed to amplify the PE and PilA gene in fusion with the NadA signal peptide at the N- terminus, ATNDDD amino acids between the pelB signal peptide and PE, a GG linker between the PE and pilA ces and a GG linker between PilA and 6xhis amino acids at the C- terminus. To e this amplification, the products of the two polymerase chain reactions describe above were used as a template with primers CAN548 and CAN535. DNA sequence corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll restriction site was incorporated into the 3’ primer. The generated PCR product was then inserted into the pET-26b(+) cloning vector (NOVAGEN®).

To generate LVL270 (M-6xHis-PE fragment-GG-PilA fragment), a polymerase chain reaction was performed to amplify the PE gene (amino acids 17-160) using the 711 vector as a template with primers CAN540 and CAN542. DNA sequence corresponding to 6xhis amino acids were incorporated into the 5’ primer (CAN540). To link the PilA sequence to the PE sequence, DNA sequence corresponding to the GG amino acids linker and the inal PilA amino acids were incorporated into the 3’ primer (CAN542). Another rase chain reaction was performed to amplify the PilA gene (amino acids 40-149 / NTHi strain 86-028NP) using pRlT16671 vector as a template with primers CAN541 and . DNA sequence corresponding to the C-terminal PE amino acids and GG amino acids were incorporated into the ’ primer (CAN541) to link the PilA to the PE sequence. Finally, to generate LVL270, a third polymerase chain reaction was performed to amplify the 6-his-PE-GG-PilA gene in fusion. To achieve this amplification, the products of the two polymerase chain reactions describe above were used as a template with s CAN540 and . DNA sequence corresponding to Nde/ restriction site was incorporated into the 5’ primer and Hind/ll restriction site was incorporated into the 3’ primer. The generated PCR product was then inserted into the pET- 26b(+) cloning vector (NOVAGEN®).

To te LVL315 (pelB signal peptide-MD-PE fragment-GG-PilA fragment-GG-6xhis), a site- directed mutagenesis was performed to change the N-terminal PE amino acid ce from QIQ to MD using LVL291 as a template with primers CAN670 and CAN671 and the QuikChange ll irected Mutagenesis Kit (Agilent Technologies, Stratagene Division).

To generate LVL317 (pelB signal peptide-PE fragment-GG-pilA fragment), a site-directed mutagenesis was performed to incorporate a stop codon between the PilA gene and the DNA sequence corresponding to GGHHHHHH amino acid residues (SEQ ID NO: 3) using LVL291 as a template with primers CAN678 and CAN679 and the QuikChange ll Site-Directed Mutagenesis Kit (Agilent logies, Stratagene Division).

To generate LVL318 (pelB signal peptide-MD-PE-GG-PilA), a site-directed mutagenesis was performed to incorporate a stop codon between the PilA gene and the DNA sequence corresponding to HH amino acid residues (SEQ ID NO: 3) using LVL315 as a template with primers CAN678 and CAN679 and the QuikChange ll Site-Directed Mutagenesis Kit (Agilent Technologies, gene Division).

To generate LVL702 (LVL291 AQ), a polymerase chain reaction was med using the LVL291 vector as template and primers CAN1517 and CAN1518. Deletion of three nucleotides corresponding to the amino acid Q at the position 23 on LVL291 ce was incorporated to the 5’ primer. The only difference between LVL702 and LVL291 is the deletion of amino acid Q at the position 23 on LVL291 sequence. NdeI and Hind/ll restriction sites were incorporated into the 5’ and 3’ primers respectively. The generated PCR product was then inserted into the pET- 26b(+) cloning vector (NOVAGEN®).

To generate LVL735 (LVL317 AQ), a polymerase chain reaction was performed using the LVL317 vector as template and primers 7 and CAN1519. Deletion of three nucleotides corresponding to the amino acid Q at the position 23 on LVL317 sequence was incorporated to the 5’ . The only difference between LVL735 and LVL317 is the deletion of amino acid Q at the position 23 on LVL317 sequence. NdeI and Hind/ll restriction sites were incorporated into the 5’ and 3’ primers tively. The generated PCR product was then inserted into the pET- 26b(+) cloning vector (NOVAGEN®).

To generate LVL736 (LVL291 + SA), a site-directed mutagenesis was performed to add amino acids 8 and A between amino acid 22 and 23 on LVL291 sequence. LVL291 was used as template with primers CAN1531 and 2 and the QuikChange ll Site-Directed Mutagenesis Kit (Agilent Technologies, gene Division).

To generate LVL737 (LVL291 + A), a site-directed mutagenesis was performed to add amino acid A n amino acid 22 and 23 on LVL291 sequence. LVL291 was used as template with primers CAN1529 and CAN1530 and the QuikChange ll Site-Directed Mutagenesis Kit (Agilent Technologies, gene Division).

To generate LVL738 (LVL291 AQIQ), a site-directed mutagenesis was performed to delete amino acids Q, | and Q at positions 23 to 25 on LVL291 sequence. LVL291 was used as template with primers CAN1523 and CAN1524 and the QuikChange ll Site-Directed Mutagenesis Kit (Agilent logies, Stratagene Division).

To generate LVL739 1 AQIQK), a irected mutagenesis was performed to delete amino acids Q, |, Q and K at positions 23 to 26 on LVL291 sequence. LVL291 was used as template with primers CAN1525 and CAN1526 and the QuikChange ll irected Mutagenesis Kit (Agilent logies, Stratagene Division).

To generate LVL740 (LVL291 AQIQKA), a site-directed nesis was performed to delete amino acids Q, I, Q, K and A at positions 23 to 27 on LVL291 sequence. LVL291 was used as template with primers CAN1527 and CAN1528 and the QuikChange ll Site-Directed Mutagenesis Kit (Agilent Technologies, Stratagene Division).

To generate LVL778 (LVL736 A6xHis tag), LVL779 (LVL737 A6xHis tag), LVL780 (LVL738 A6xHis tag), LVL781 (LVL739 A6xHis tag) and LVL782 0 A6xHis tag) a polymerase chain reaction was performed using the LVL736, LVL737, LVL738, LVL739 and LVL740 s as template, respectively, with primers CAN1669 and CAN543. Deletion of 6xHis tag corresponds to the amino acid sequence GGHHHHHH (SEQ ID NO. 3) at the C-terminal sequences. This deletion was incorporated to the 3’ primer. NdeI and Hind/ll restriction sites were incorporated into the 5’ and 3’ primers respectively. The generated PCR product was then inserted into the b(+) cloning vector (NOVAGEN®).

Table 4: PCR primer sequences used for PE, PilA and PE-PilA ications DNA Sequence '—3' CACACACATATGATTAAATTTCTCTCTGCATTAATTCTTCTACTGGTCACGACGG CAN534 CGGCTCAGGCTGAGACTAAAAAAGCAGCGGTATCTG (SfiQ 3 NO. 155) TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTGTGTGACACTTCCG CAN535 CAAAAATTTGC (SfiQ 3 NO. 156) TTTGCGGAAGTGTCACACAAGGCGGCGCGCAGATTCAGAAGGCTGAACAAAATGA CAN536 TGT (SfiQ 3 NO. 157) TTTTGTTCAGCCTTCTGAATCTGCGCGCCGCCTTGTGTGACACTTCCGC CAN537 AAA (SfiQ 3 NO. 158) TGTGTGAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCCTTTTTTATCAACTGAA CAN538 AATG (SfiQ 3 NO. 159) CACACACATATGCACCACCACCACCACCACAGCGCGCAGAT TCAGAAGGCTGAAC CAN540 ATGT (SiiQ :3 NO. 160) CATTTTCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATC (s '.

CAN541 NO. 161) GATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAAATG (s: CAN542 NO. 162) CAN543 TGTGTGAAGCTTTTATTGTGTGACACTTCCGCAAA (S '. 3 NO.

CATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCG CTGCCCAGCCGGCGATGGCCCAGATTCAGAAGGCTGAACAAAATGATGT (SI CAN544 __D NO . l 64) TCAGTTGATAAAAAAGGCGGCACTAAAAAAGCAGCGGTATCTG (s: CAN545 :3 NO. 165) CAGATACCGCTGCTTTTTTAGTGCCGCCTTTTTTATCAACTGAAAATGC (s: CAN546 :3 NO. 166) CACACACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCG CTGCCCAGCCGGCGATGGCCGATATTCAGAAGGCTGAACAAAATGATGT (SEQ CAN547 __D NO . l 67) CACACACATATGAAACACT T TCCATCCAAAGTACTGACCACAGCCATCCT TGCCA CTTTCTGTAGCGGCGCACTGGCAGCCACAAACGACGACGATAAGGCTGAACAAAA CAN548 TGATG (SfiZQ :3 NO. 168) CAN678 GGAAGTGTCACACAATAAGGCGGCCACCACCACC (S 4'.Q 3 NO. 2-71) CAN679 GGTGGTGGTGGCCGCCTTATTGTGTGACACTTCC (S 4'.Q 3 NO. 2-72) GATATACATATGAAATACCTGCTGCCGACCGCTGCTGCTGGTCTGCTGCTCCTCG CAN1517 CTGCCCAGCCGGCGATGGCCATTCAGAAGGCTGAACAAAA(S*'.Q 3 NO. 205) CAN1518 GGCCGCAAGCTTTTAGTGGTGGTGGTGGTGGTGGCCGCC(S*'.Q 3 NO. 206) CAN1519 GGCCGCAAGCTTTTATTGTGTGACACTTCC(S*'.Q 3 NO. 207 ) GCTGCCCAGCCGGCGATGGCCAAGGCTGAACAAAATGATGTG (8de :3 NO.

CAN1523 208) CACATCATTTTGTTCAGCCTTGGCCATCGCCGGCTGGGCAGC (s: CAN1524 209) GCTGCCCAGCCGGCGATGGCCGCTGAACAAAATGATGTGAAGC CAN1525 2 l 0) WO 39225 GCTTCACATCATTTTGTTCAGCGGCCATCGCCGGCTGGGCAGC CAN1526 2 :- 1) GCTGCCCAGCCGGCGATGGCCGAACAAAATGATGTGAAGCTGG CAN1527 2 3—2) CCAGCTTCACATCATTTTGTTCGGCCATCGCCGGCTGGGCAGC 8 2 l 3 ) GCTGCCCAGCCGGCGATGGCCGCCCAGATTCAGAAGGCTGAAC 9 2 l 4) GTTCAGCCTTCTGAATCTGGGCGGCCATCGCCGGCTGGGCAGC (S '.

CAN1530 215) GCTGCCCAGCCGGCGATGGCCAGCGCCCAGATTCAGAAGGCTGAAC CAN1531 NO. 216) GTTCAGCCTTCTGAATCTGGGCGCTGGCCATCGCCGGCTGGGCAGC (S'.

CAN1532 NO- 217) CAN1669 CACACACATATGAAATACCTGCTGCCGACC (S '. 3 NO. 218) GAATTCCATATGCACCATCACCATCACCATACTAAAAAAGCAGCGGTATCTGAA MDesPILA- (S41Q 3 NO. 173) MDesPILAGCGCCGCTCGAGTCATTGTGTGACACTTCCGC (S '. 3 NO.

GCCCAGCCGGCGATGGCCCAGATCCAGAAGGCTGAACAAAATG MnoNTHL (s: 44 175) CATTTTGTTCAGCCTTCTGGATCTGGGCCATCGCCGGCTGGGC MnoNTHL (s: 45 176) Transformation ichia coli BLR (DE3) or E. coli HMS (DE3) cells were ormed with plasmid DNA according to standard methods with CaClz-treated cells. (Hanahan D. « Plasmid transformation by Simanis. » ln Glover, D. M. (Ed), DNA cloning. IRL Press London. (1985): p. 109-135.).

Briefly, BLR (DE3) or HMS174(DE3) competent cells were gently thawed on ice. Approximately 4pl of plasmid (10-100 ng) were mixed using 50-100 pl competent cells. Thereafter, this formulation was incubated on ice for 30 min. To perform the transformation reaction, the formulation was heat pulsed at 42°C for 45 seconds then incubated on ice for 2 minutes.

Approximately 0.5 ml of SOC medium (Super l broth with lite repression) was added to the transformed cells and the cell culture was incubated at 37°C for one hour before plating on Luria-Bertani (LB) agar with 50 ug/ml kanamycin. Around 100 pl of ormed cell culture was plated and incubated overnight at 37°C.

BLR (DE3): BLR is a recA‘ derivative of BL21 (F— ompT hstB(rB— mB—) gal dcm (DE3). This E. coli strain used for expression of recombinant proteins improves plasmid monomer yields and may help stabilize target plasmids containing repetitive sequences or whose products may cause the loss of the DE3 prophage. er, F.W. (1991) J. Mol. Biol. 219: 37—44). The detailed genotype of E.coli BLR (DE3) has been published by N®. (F- ompT hstB (rB- mB-) gal dcm recA)306::Tn10 (TetR) (DE3).

HMS174 (DE3): HMS174 s provide the recA mutation in a K—12 background. Like BLR, these s may stabilize certain target genes whose products may cause the loss of the DE3 prophage. The detailed genotype of E.coli HMS174 (DE3) has been published by NOVAGEN®.

(F— recA1 hst(rK12— mK12+) (DE3) (Rif R ).

Production using BLR (DE3) and Characterization of His tagged constructs are described in Example 3 through Example 6 Example 3: Protein expression using shake flask Generally, one confluent agar plate inoculated with Escherichia coli BLR (DE3) transformed with recombinant plasmid was stripped, resuspended in culture media and used to inoculate 800 ml of LB broth (Becton, Dickinson and Company) 1r 1% (weight/volume, w/v) glucose (Laboratoire MAT, catalogue number: GR-0101) and 50ug/ml kanamycin (Sigma) to obtain O.D.600nm between 0.1 and 0.2. Cultures were incubated at 37 °C with agitation of 250 RPM to reach an 0nm Of ~0..8 One ml of each culture was then collected, centrifuged at 14 000 RPM for 5 minutes and supernatants and pellets were frozen at -20°C separately.

At an O.D.600nm ~0.8, the BLR (DE3) cultures were cooled down (-20°C, 20 minutes or 4°C, 1 hour, preferably at 4°C for 1 hour) before inducing the expression of the recombinant protein by addition of 1 mM isopropyl B-Dthiogalactopyranoside (IPTG; EMD Chemicals Inc, catalogue number: 5815) and incubation overnight at 16, 22 and 30°C, or 3 hours at 37°C with agitation of 250 RPM, preferably overnight at 22°C. After the induction period the cultures were centrifuged at 14 000 RPM for 5 minutes or 6 000 RPM for 15 minutes and supernatant (media on sample) and pellets ining soluble and insoluble fractions) were frozen at -20°C separately.

These ions are used for periplasmic protein expression.

Example 4: Protein purification using shake flask, cell pastes, His tagged constructs Each bacterial pellet obtained after induction was resuspended in 20 mM ydroxyethyl) piperazineethanesulfonic acid ) buffer (pH 8.0) ning 500 mM NaCl, 10 mM imidazole and Roche COMPLETE® Protease tor Cocktail (1 tablet/50 ml of HEPES buffer containing 500 mM NaCl, Roche COMPLETE® ULTRA tablets, Roche Diagnostics Corporation).

Alternatively, 20 to 50 mM bicine buffer may be used instead of HEPES buffer containing NaCl.

For example, 20 mM bicine buffer may be used. Bacteria were lysed using a Constant System 1.1 KW 2 X 30 000 PSI (pounds per square inch). Soluble natant) and insoluble (pellet) components were separated by centrifugation at 20 000g for 20 min at 4°C. 6-His tagged-proteins were purified under native ions on immobilized metal affinity chromatography (IMAC) using PROFINIATM protein purification protocol (Bio-Rad Laboratories, Inc.) The soluble components were loaded on a 5m| His Trap column (Bio-Rad Laboratories, Inc.) preequilibrated with the same buffer used for ial resuspension; the soluble components were added at up to 5 ml/min (producing a “flow through fraction”) After loading on the column, the column was washed with 10 column volumes of the same buffer at a rate of 10 ml/min (producing a “wash fraction #1). A second wash using 20 mM bicine buffer or 20 mM HEPES buffer (pH 8.0) containing 500 mM NaCl and 20 mM imidazole was performed, producing a “wash fraction #2). Elution was med using 2 column volumes of 20mM HEPES buffer or 50mM bicine buffer (pH 8.0) containing 500 mM NaCl and 250 mM imidazole at a rate of 10 ml/min, ing an “elution fraction”.

To improve the purity of the n, positive elution fractions from IMAC were pooled and loaded on a size exclusion chromatography (SEC) column DTM SUPERDEXTM 200 26/60 from GE Healthcare) preequilibrated in phosphate buffered saline without calcium or magnesium (NaCl 137 mM, KCI 2.7 mM, NazHPO4 8.1 mM, KH2PO4 1.47 mM, pH 7.4). s from elution ons were ed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Samples were trated using Centricon 10 000 MW (Millipore).

Protein concentration was determined using spectrometer.

Example 5: SDS-PAGE and Western Blot Analzsis of His tagged constructs & SDS-PAGE is of non-his tagged LVL317 & LVL318 constructs e and insoluble fraction preparation For example, 1 ml of culture after induction (see, for example, Example 3 above) was centrifuged at 14 000 RPM for 2 min. The pellet was resolubilized using 40 pl of BUGBUSTER® Protein Extraction Reagent (NOVAGEN®, EMD4 Biosciences, Merck), creating a cell suspension. The cell suspension was incubated on a rotating platform for 10 min at room temperature. The cell suspension was then centrifuged at 14 000 RPM for 2 min to separate the soluble fraction. The resulting pellet (insoluble fraction) was resolubilized using 70 pl of deionized water, 5 pl of dithiothreitol (DTT) 1M and 25 pl of NUPAGE® LDS (Lithium Dodecyl Sulphate) Sample Buffer 4X (INVITROGENTM). The soluble fraction (supernatant from the cell suspension of the resolubilized pellet) was added to 30 pl of deionized water, 5 pl of DTT 1M and 25 pl of LDS Sample Buffer 4X.

Media on preparation For example, to prepare the media fraction, 100 pl of the atant from the induced whole cell culture following centrifugation (see, for example, Example 3 above) was concentrated by adding 500 pl of RC reagent l (Bio-Rad Laboratories, Inc); the sample was mixed and incubated for 1 min at room ature. Then, 500 pl of Reagent ll (Bio-Rad Laboratories, Inc.) was added to the sample and mixed. This formulation was centrifuged at 14 000 RPM for 10 min. The pellet was resolubilized using 28 pl of deionized water, 2 pl of DTT 1M and 10 pl of LDS SB 4X.

Purification fraction preparation For example, purified proteins (for example, obtained as described in Example 4) were prepared for SDS-PAGE analysis by adding 70 pl of sample, 5 pl of DTT 1M and 25 pl of LDS Sample Buffer 4X.

SDS—PAGE analysis and transfer to nitrocellulose membrane SDS—PAGE analysis and transfer to nitrocellulose membrane were performed according to manufacturer’s endations (lnvitrogen) using NUPAGE® Bis-Tris 4-12% gels.

Preparations of samples, buffers and migration conditions were done under conditions recommended by the ers.

In one example, the gel was loaded with a 20 ul sample from a master mix comprising 70 pl of a ed protein fraction, 5 pl of DTT 1M and 25 pl of LDS SB 4X.

After samples were run on NUPAGE® Bis-Tris 4-12% gels, the proteins were transferred to nitrocellulose membranes.

Nitrocellulose membranes were d for 30 minutes at 37°C, 60 RPM using 3 % milk/ PBS 1X fresh solution. After the ng incubation, Primary Antibodies were added (6X His Tag® antibody, Abcam PLC, catalogue number: ab9108) at a dilution of: 1:1000 in 3 % milk/ PBS 1X fresh solution for 1 hour at 37°C, 60 RPM. After that, membranes were washed three times, for minutes each, at room temperature using 0.02% polsorbate 20 (for example, TWEENTM 20) / PBS 1X. Secondary Antibodies ine phosphatase (AP) Rabbit anti-IgG (H+L) rabbit, Jackson Research Laboratories, Inc.) were added at dilution 1:14 000 using 3 % milk/ PBS 1X fresh solution. Membranes were incubated for 1 hour at 37°C, 60 RPM. After that, membranes were washed three times for 5 s at room ature using 0.02% rbate 20 (for example, TWEENTM 20) / PBS 1X before the membrane expositions to 5- bromochloro-3—indolyl phosphate/nitro blue tetrazolium (for example, BCIP®lNBT from Sigma- Aldrich®, 1 tablet/ 10 ml water).

See Figure 1 for SDS-PAGE of induced bacterial extracts for fusion protein constructs LVL291, LVL268 and LVL269. Insoluble fraction (I), Soluble fraction (S) and Culture Media fraction (M) were loaded for LVL291, LVL268 and LVL269 before and after induction (ind).

See Figure 2 for SDS-PAGE and Western blot related to purification extracts for fusion n constructs LVL291, LVL268 and LVL269. Flow through fraction (Ft), Wash fraction (W) and Elution fraction (E) were loaded for purification of LVL291, LVL268 and . Anti-his tag was used to probe extracts.

See Figure 3 for SDS-PAGE of induced ial and purification extracts for fusion protein constructs LVL291 and LVL315. Culture Media fraction (M), Soluble on (Sol), Insoluble on (Ins), Flow through fraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution on (E) were loaded for LVL291 and LVL315.

See Figure 4 for SDS-PAGE of induced bacterial and purification extracts for fusion protein construct LVL312. Culture Media fraction (M), Soluble fraction (Sol), Insoluble fraction (Ins), Flow Through fraction (Ft), Wash fraction #1 (W1), Wash fraction #2 (W2) and Elution fraction (E) were loaded for LVL312.

See Figure 25 for SDS-PAGE of soluble fractions from induced bacterial extracts for fusion protein constructs LVL291, LVL702, LVL736, LVL737, LVL738, LVL739, LVL740 and pET26b vector (negative control). (a) ment 1 (b) Experiment 2 (c) Experiment 3. PE-PilA fusion protein indicated by arrow.

See Figure 26 for the e band percentage of fusion protein in the soluble fraction from Experiments 1, 2 and 3.

LVL317 and LVL318 bacterial extracts used in the SDS—PAGE analysis in Figure 5 and Figure 6, respectively, were ed generally as described above.

Figure 5. SDS-PAGE of induced (1mM and 10uM IPTG) bacterial extracts for fusion protein construct LVL317. ts from before (NI) and after induction (In), Soluble fraction (S), Insoluble fraction (I).

Figure 6. SDS-PAGE of induced (1mM and 10uM IPTG) bacterial extracts for fusion protein construct LVL318. Extracts from before (NI) and after induction (In), Culture Media fraction (M), Soluble fraction (S), Insoluble fraction (I).

Proteins separate by SDS-PAGE were transferred to an lmmobilon-P membrane. The Coomassie Blue stained protein bands were cut and placed in a sequenator reactor. cing was carried out ing to manufacturer’s protocol using an Applied tems PROCISE® Protein Sequencer, model 494-cLC.

Table 5: Shake flask protein expression profiles and signal peptide cleavage for fusion protein constructs.

Fusion ption Protein Signal Protein N-term —> C-term Expression peptide Construct profile cleavage F|g| sp — E — PiIA fragment — GG — PE fragment — LVL312 med GGHHHHHH LVL291 Confirmed PeIB sp — D — PE fragment — GG — PiIA fragment — LVL268 Confirmed GGHHHHHH NadA sp — ATNDDD — PE fragment — GG — PiIA fragment — LVL269 Confirmed GGHHHHHH LVL270 MHHHHHH — PE fragment — GG — PiIA fragment : Not tested PeIB sp — MD — PE fragment — GG — PiIA fragment — LVL315 Confirmed GGHHHHHH LVL317 PeIB — PE fragment — GG — PiIA fragment : Confirmed LVL318 PeIB sp — MD — PE fragment — GG — PiIA fragment LVL702 PeIB sp — PE fragment — GG — PiIA fragment — GGHHHHHH : Confirmed LVL736 PeIB sp — PE fragment — GG — PiIA nt — GGHHHHHH : Confirmed LVL737 PeIB sp — PE fragment — GG — PiIA fragment — GGHHHHHH : Confirmed LVL738 PeIB sp — PE nt — GG — PiIA fragment — GGHHHHHH : Confirmed LVL739 PeIB sp — PE nt — GG — PiIA fragment — GGHHHHHH : Confirmed LVL740 PeIB sp — PE fragment — GG — PiIA fragment — GGHHHHHH : Confirmed So = Soluble fraction. In = Insoluble fraction. Se = Protein Secreted in the media fraction. Nt = Not tested. The ing rating were based on a visual inspection (coomassie) + : low expression; ++ : medium expression; +++ : high expression; - : no sion Exam Ie 6: LVL291 Fusion rotein characterization PHYSICAL PROPERTIES OF LVL291: Foldin of PE and Pi/A in LVL291 & Melt/n Point Circular Dichromism .' Analysis of Secondary Structure Circular dichroism (CD) is used to determine the secondary structure composition of a protein by measuring the difference in the tion of left-handed polarized light versus right-handed polarized light which is due to structural asymmetry. The shape and the magnitude of the CD spectra in the far-UV region (190-250nm) are different whether a protein exhibits a beta-sheet, alpha-helix or random coil structure. The relative abundance of each secondary structure type in a given protein sample can be calculated by comparison to reference spectra.

Far UV spectra are measured using an l path of 0,01cm from 178 to 250nm, with a 1nm resolution and bandwidth on a Jasco J-720 spectropolarimeter. Temperature of the cell is maintained at 23°C by a Peltier thermostated RTE-111 cell block. A nitrogen flow of 10L/min is ined during the measurements.

Results: The far-UV CD spectra obtained for PE (from construct pRlT16762), PilA (from construct pRlT 16790) and PE-PilA proteins are characteristic of folded proteins containing a mix of alpha and beta structures, but PE is significantly richer in alpha helix than PilA and PE-PilA (Figure 7, CD spectra of PE, PilA and PE-PilA fusion proteins).

In order to evaluate the integrity of the folding of PE and PilA individual ns once bound together in a chimeric protein and then verify a possible interaction between both, difference a were calculated.

. When the PE and PilA far-UV spectra are combined, the resulting spectrum superposes to the um of PE-PilA chimer (Figure 8, Combination of PE and PilA CD um).

This result suggests that the PE-PilA chimer contains all the secondary structures that are detected in the individual components. It also suggests that the fusion of the ns has no major impact on the ary ures of the dual components and consequently that the folding of PE and PilA is not significantly different whether the proteins are separate or in fusion.

Melting Point Evaluation: In order to evaluate if the expression in fusion has an impact on the thermodynamic properties of the individual proteins, the melting points of PE, PilA and PE-PilA have been evaluated by monitoring the defolding of the alpha helix with temperatue by circular ism.

The presence of alpha helix is characterized by a m in the Circular dichroism signal at 222nm, so a significant increase in CD signal at 222nm during temperature increase is an indication of protein denaturation. The determination of the temperature at which the protein undergoes loss in secondary structure allows the determination of the melting point (Tm), which corresponds to the temperature at which half of the proteins have lost their structure.

Melting point can be determined by identification of the inflexion point on the l denaturation curve obtained from a temperature versus CD 222nm plot.

. Melting point of PilA and PE as determined by far-UV CD are respectively of 52°C and 68°C (Figure 9, PilA thermal denaturation curve; Figure 10, PE thermal ration curve).

. The PE-PilA fusion protein exhibits two distinct Tm’s at 48°C and 71°C (Figure 11, PE- PilA fusion protein thermal denaturation curve). Those values te that the PE and PilA proteins are still independently folded when bound into a chimer and that they defold at a similar temperature whether they are separate or in fusion. The ation that the defolding of the PilA portion at 48°C doesn’t cause precipitation or impact the Tm of the PE portion at 71°C is a strong indication that the interaction between PE and PilA within the fusion is minimal and that they don’t have a major observable impact on each other. The melting points of proteins are sensitive to various external conditions, including buffer ition or presence of interacting molecules; that no major ion is observed upon fusion of PE and PilA is a strong indication of the preservation of most of the structure and of the properties of both PE and PilA when they are bound together.

Example 7: Fermentation process Fusion proteins of the invention may be prepared by methods known by those skilled in the art.

Example 8: Protein Purification of PEI PiIAI and LVL317 PE protein purification from pR/T16762: To generate the 762 expression , the pRlT16711 vector was digested using BamHI and Ncol restriction enzymes in order to delete 6 amino acid residues between the signal sequence (pelB) and PE. The vector obtained was named pRlT16712. In this vector, there are 3 amino acids between the signal sequence pelB and PE: MDP. In a second step, a site directed nesis was performed to change amino acid sequence from MDP to QIQ using pRlT16712 as template with primers MnoNTHi-44 and MnoNTHi-45 (described in Table 4) and the QuikChange ll irected nesis Kit (Agilent Technologies, Stratagene Division).

Working seed of E. coli BLR(DE3) containing PE QIQ (from the pRlT16762 construct) was thawed from -80°C and used to prepare 100 ml of pre-culture in LB broth by overnight incubation at 37°C under agitation at 215 RPM. After overnight incubation, eight flasks containing 800 ml of LB APS were inoculated with 12.5 ml of pre-culture and ODeoo measured at around 0.06. The cultures were incubated 3h at 37°C with shaking. At a ODeoo of around 0.9, 1mM IPTG was added to start the induction. During the induction, the cultures were incubated 19h at 22°C with shaking. After induction, ODeoo was at around 2.2. The cell cultures were transferred into 1L centrifuge bags placed inside 1L s and centrifuged at 4°C for 30 minutes at 6,000xg and atant discarded. 1m| aliquots of culture pre- and post-induction and supernatant were kept for future is.

Lysis of the BLR(DE3) induced with PE QIQ The centrifuge bags were d from the centrifugation bottles, opened and the pellet was expulsed from the bag into a beaker. The eight pellets were pulled together and resuspended in 100ml of binding buffer (20mM Hepes, 10mM imidazole, 500mM NaCl, pH 8.01). The Eco/i BLR (DE3) ning the PE QIQ contruct were disrupted with the TS Series Bench Top cell disrupter from Constant s Ltd. (1x30 szi; 1x15szi). The lysate was centrifuged 30 minutes, 6000RPM, 4°C. The supernatant was kept and loaded on an IMAC column.

IMAC purification of PE QIQ IMAC column (BioRad, Bio-Scale Mini Profinity IMAC cartridge 5ml) was brated with 5CV of Binding buffer (20mM HEPES, 10mM imidazole, 500mM NaCl, pH 8.01) at 5ml/min. 100ml of lysate supernatant was loaded on the IMAC at 2.5mL/min. Flow-through was collected in 50ml fractions for future analysis. The column was washed with 3CV of Binding buffer to remove unbound protein. Sample containing unbound proteins was collected in one aliquot of ml in a 50 ml tube. The column was washed with 2CV of Wash buffer (20mM HEPES, 20mM imidazole, 500mM NaCl, pH 8.01) collected in 2 ml fractions in a 96 well plate. The bound protein was then eluted with 6CV of 100% Elution buffer (20mM HEPES, 250mM imidazole, 500mM NaCl, pH 8.01). The eluted protein was collected in 2 ml fractions in 96- well . Wash and elution were performed at n.

Size exclusion chromatography (SEC) on the IMAC pool of PE QIQ SEC column (GE healthcare, HILOADTM 26/60 SUPERDEXTM 75 prep grade, 60cm height approx 319ml volume) was equilibrated with 3CV of SEC buffer (20mM HEPES, 150mM NaCl, pH8.49). 11 ml of IMAC eluate was loaded onto the column at a flow rate of 2.5 . 2m| fractions were collected from 0.3CV to 0.9CV. Two runs were performed then fractions were analyzed by SDS-PAGE. Fractions from the two runs ning Prot E protein were pooled together (“SEC pool” 48ml approx total volume). 500mM of ne was added to the SEC pool.

Dosage of the PE QIQ poo/ed samples generated in the above SEC protocol The SEC pool was dosed with the RCDC (Reducing Agent and ent Compatible) method from the Bio-Rad RC DCTM kit following manufacturer’s protocol: For each tested sample and standard, 25uL was distributed in uge tubes in duplicate. 125uL of d RC Reagent l was added into each tube; each tube was vortexed and incubate for 1 minute at room temperature. 125uL of Bio-Rad RC Reagent II is added into each tube; each tube is ed and then centrifuged at 14,000xg for 5 minutes.

Supernatants are discarded by inverting the tubes on clean, adsorbent tissue paper allowing the liquid to drain completely from the tubes. 25.4uL of Reagent A (already prepared by mixing 20uL of Reagent S per 1ml of Reagent A) is added to each tube; each tube is vortexed and incubated at room temperature for 5 minutes, or until itate is completely dissolved.

Vortex before proceeding to next step. Add 200uL of DC reagent B to each tube and vortex immediately. Incubate at room temperature for 15 minutes. Transfer all samples to a l plate and read the adsorbance at 750nm to determine the protein tration for each unknown protein sample.

The ProtE concentration was 1.069 mg/ml PilA His-tagged protein purification: PilA was purified following the general procedure below: E. coli cells containing a construct encoding PilA or a fragment thereof are suspended in BUGBUSTER® and BENZONASE® nuclease (NOVAGEN®), for example 10 ml BUGBUSTER® and 10 ul BENZONASE® nuclease. The cell lysate is mixed at room temperature on a rotating platform, for example, for 15 s. The cell lysate is centrifuged at 4°C, for example at 16,000g for 20 minutes. The supernatant containing the protein is added to a Ni NTA column containing Ni NTA HIS -B|ND® resin and mixed at 4°C, for example for 1 hour. The column may consist of2 ml of Ni NTA HIS -B|ND® resin (NOVAGEN®) and 10 ml 1X Binding Buffer (from NOVAGEN®’s Ni-NTA Buffer Kit). The column flow through is then collected. The resin is washed two times with 1X wash buffer, for example, containing 300 mM NaCl, 50mM NaHzPO4, 25 mM imidazone, pH 8.0). The wash is ted by gravity flow. The protein is eluted from the column with 1X elution buffer, for example, 300 mM NaCl, 50mM NaHzPO4, 250 mM imidazone, pH 8.0. The protein may be further purified by dialysis with the Binding Buffer and rerun over a Ni NTA column as described above.

Thrombin cleavage of PilA.

PilA is then incubated with thrombin (diluted 1/50) at room temperature for 16h, to remove the histidine tag.

Size exclusion chromatography (SEC) on Pi/A cleaved with thrombin.

SEC column (GE healthcare, HILOADTM 26/60 EXTM 75 prep grade, 60cm height approx 319ml volume) was equilibrated with 5CV of SEC buffer (20mM HEPES, 150mM NaCl, pH8.52). Approximately 10 ml of cleaved PilA was loaded onto the column at a flow rate of 2.5 ml/min. 2m| fractions collected from 0.3CV to 0.9CV. Two runs were med then ons were analyzed by SDS—PAGE. Fractions from the two runs ning cleaved PilA protein were pooled together (“SEC pool”, 52ml approx total ).

Dosage of PilA, SEC pool.

The SEC pool was dosed with the RCDC method as described above. The cleaved PilA concentration was at 5.37 mg/ml.

Dialysis of the PilA SEC pool with PBS 1x pH 7.4 (dialysis factor = 1600) and dosage by RCDC The concentration post-dialysis determined by RCDC was at 3.0 mg/ml.

Purification of L VL31 7 Osmotic shock Since LVL317 fusion protein is expressed and processed in bacterial asm, the protein was extracted by osmotic shock.

Frozen (-20°C) harvested E. coli B2448 cell paste containing LVL317 from 4 L of fermentor culture were pooled and resuspended in a hypertonic buffer consisting of 24 mM Tris-HCl, 16% (w/v) sucrose, 9.9% (w/v) glucose, 10 mM EDTA, pH 8.0 up to a final volume of 4L. The suspension was mixed gently for 30 min at room ature using a e propeller installed on RW 16 basic stirrer, at medium speed. The suspension was centrifuged at 15,900 x g for 30 minutes at room temperature. Supernatant (SN1) was kept for gel analysis.

The resulting pellet was resuspended in a hypotonic solution; 38 mM MgClz, and mixed for 30 min at room temperature. The mixture was centrifuged at 15,900 x g for 30 minutes at room temperature and the n recovered in the supernatant (SN2).

A clarification of the SN2 was performed by tion through a 0.45/0.2 um polyethersulfone Sartorius Sartopore 2 MidiCap filter, at 600ml/min of flow rate.

The SN2 was d 1:3 with 20 mM NaHzPO4-Na2HPO4, pH 7.0, the pH adjusted to 7.0 if necessary and another clarification by filtration h a 0.45/0.2 um polyethersulfone Sartorius Sartopore 2 MidiCap filter, at 600ml/min was performed.

SP SEPHAROSETM Fast Flow (SP FF) chromatography The diluted/filtered SN2 was loaded and captured on a strong ic exchanger resin (SP SEPHAROSETM FF - GE Healthcare) in a 14 cm ID (internal diameter) x 20 cm length column (column volume 3100ml) equilibrated with 2CV of 20 mM NaHzPO4/ NazHPO4 buffer pH 7.0.

After washing the column with 5CV of 20 mM NaHzPO4 / NazHPO4 buffer pH 7.0, the antigen ined within LVL317) was eluted by increasing the concentration of NaCl up to 100 mM in the same washing buffer.

See Figure 12 for a typical SP SEPHAROSETM Fast Flow chromatogram.

Q SEPHAROSETM Fast Flow (Q FF) chromatography The antigen t in the SP FF Eluate was diluted 1:4 with a 20 mM Tris pH 8.5, pH adjusted to 8.5 if ary and passed through a strong anionic exchanger resin (Q SEPHAROSETM FF - GE Healthcare) in a 14 cm ID x 11.8 cm length column (column volume ) equilibrated with 2CV of 20 mM Tris buffer pH 8.5. The antigen was recovered in the flow-through fraction.

See Figure 13 for a typical Q SEPHAROSETM Fast Flow chromatogram.

Concentration, diaflitration, rbate 80 addition and sterile filtration The Q FF flow-through containing the antigen was trated up to 0.7-0.8mg/ml based on chromatogram UV and diafiltered with 5DV of 10 mM KHZPO4 / KZHPO4 buffer pH 6.5 using a Pellicon-2TM 10 kDa cutoff membrane (Millipore).

Using a 5% stock solution, polysorbate 80 (for example, TWEENTM 80) was added to the ultrafiltration retentate and agitated for 30 minutes with magnetic r at 130rpm at 4°C. The final concentration of polysorbate 80 was 0.04%. Ultrafiltration retentate was sterilized by filtration through a 0.45/0.2 um Cellulose Acetate membrane (Sartobran 300, Sartorius). The purified bulk was stored at —20°C or -80°C. Absolute protein concentration was measured by AAA (Amino Acid is) at 0.737mg/ml.

Example 9: Use of Polysorbate 80 A titration experiment indicated that the addition of polysorbate 80, specifically, TWEENTM 80 to a final concentration of 0.04% (w/v) to the purified bulk prior to sterile filtration reduced filamentous particle formation and aggregation.

According to DSC analysis, TWEENTM 80 d the degree of structural change °C) seen after freeze/thaw cycles after storage at -20°C and after storage 4 days at 4°C, -20°C and -80°C and 37°C. e 10: SDS-PAGE and Western Blot is of LVL317 SDS-PAGE and Western Blot analysis: NUPAGE®, Bis-Tris 4-12% gel was loaded as described below with 10ug of sample in NUPAGE® LD sample buffer ning 50mM DTT heated 5min at 95°C (20uL of sample was loaded for samples having low concentration). Migration: 35 minutes at ts at room temperature (RT) in NUPAGE( MES Running Buffer. Gel Stained 2 hours in Instant blue (Novexin cat.: ISBO‘IL) and destained WO 39225 overnight in water.

Lane contents: 1: MW standard (10uL) 2: Start (total fraction) (10ug) 3: SN1 non filtered (10ug) 4: SN2 not ed (10ug) 5: Not extracted (10ug) 6: Load SP FF (10ug) 7: Flow through SP FF (6.9ug) 8: Wash SP FF (20uL) 9: Elution SP FF (10ug) : Strip SP FF (10ug) 11: Load Q FF ) 12: Elution Q FF (9.8ug) 13: Strip Q FF (4.8ug) 14: TFF retentate before0.04% TWEENTM 80 spiked (10ug) : Purified bulk Not filtered 0.04% TWEENTM 80 spiked (10ug) 16: Purified bulk e Filtered 0.04% TWEENTM 80 spiked (10ug) 17: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (20ug + spiked E. Coli Cell lysate Rix (H— 18: E. Coli Cell lysate Rix (2ug) 19: E. Coli Cell lysate Rix (1 pg) : E. Coli Cell lysate Rix (0.5ug) See Figure 14 for a SDS-PAGE of ln-process samples from purification process of PE-PilA fusion protein.

For Western Blot, proteins were transferred at 4°C overnight at 30Volts in NUPAGE® transfer buffer + 20% Methanol, 0.1% SDS on nitrocellulose membrane. Membranes were blocked 1 hour with 50mM Tris, 150mM NaCl pH 7.4 + 5% non—fat dry milk, incubated 2 hours in rabbit onal primary antibody diluted in blocking buffer (anti-Prot—E 1/50 000 and anti-Ecoli (BLR) 1/1 000), washed 3x5minutes in 50mM Tris pH 7.4 + 0.05% Tween 20, incubated 1 hour in secondary antibody (goat anti-rabbit conjugated to alkaline phosphatase diluted 1/5000 in ng buffer), washed 3x5minutes in wash buffer and ped in BClP/NBT ate (1 tablet per 10ml). All incubations performed in 25ml per membrane.

See Figure 15 for a Western Blot of ln-process samples of cation process from PE-PilA fusion protein. Blot using rabbit polyclonal anti-PE.

Lane contents: 1: MW standard (10uL) 2: Start (total on) (10ug) 3: SN1 non filtered (10ug) 4: SN2 not filtered (10ug) 5: Not extracted (10ug) 6: Load SP FF (10ug) 7: Flow through SP FF (6.9ug) 8: Wash SP FF (20uL) 9: Elution SP FF (10ug) : Strip SP FF (10ug) 11: Load Q FF (8.9ug) 12: Elution Q FF (9.8ug) 13: Strip Q FF (4.8ug) 14: TFF retentate before0.04% TWEENTM 80 spiked (10ug) : Purified bulk Not filtered 0.04% TWEENTM 80 spiked (10ug) 16: Purified bulk Sterile ed 0.04% TWEENTM 80 spiked (10ug) 17: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (20ug + spiked E. Coli Cell lysate Rix (H— 18: E. Coli Cell lysate Rix (2ug) 19: E. Coli Cell lysate Rix (1 pg) : E. Coli Cell lysate Rix (0.5ug) See Figure 16 for a Western Blot of cess samples of purification process from A fusion n. Blot using rabbit polyclonal anti-Eco/i (BLR).

Lane contents: 1: MW standard (10uL) 2: Start (total fraction) (10ug) 3: SN1 non ed (10ug) 4: SN2 not filtered (10ug) 5: Not extracted (10ug) 6: Load SP FF (10ug) 7: Flow through SP FF (6.9ug) 8: Wash SP FF (20uL) 9: Elution SP FF (10ug) : Strip SP FF (10ug) 11: Load Q FF (8.9ug) 12: n Q FF ) 13: Strip Q FF (4.8ug) 14: TFF retentate before0.04% TWEENTM 80 spiked (10ug) : Purified bulk Not filtered 0.04% TWEENTM 80 spiked (10ug) 16: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (10ug) 17: Purified bulk Sterile Filtered 0.04% TWEENTM 80 spiked (20ug + spiked E. Coli Cell lysate Rix (H— 18: E. Coli Cell lysate Rix (2ug) 19: E. Coli Cell lysate Rix (1 pg) : E. Coli Cell lysate Rix (0.5ug) SDS-PAGE and Western Blot figures comments: The PE-PilA fusion protein migrates at 30kDa.

The extraction by c shock extracts the fusion protein expressed and processed in bacteria periplasm and reduced contamination from bacteria. Small loss of fusion protein during hypertonic treatment (lane 3). A small proportion is not extracted by hypotonic treatment and s associate with cells (lane 5). Small loss in SP FF Flow through (lane 7) and in strip fraction of both columns (lanes 10 and 13). Since the total volume of strip fraction is low the loss of fusion protein is not significant. Degraded bands are visible in strip fractions but not in final product. No significant contamination from E. coli host cell proteins in purified bulk (lane 16).

Analysis of LVL735 and LVL778 yielded similar profiles as LVL317. e 11 : Melting Point Data for PE, FHA and LVL317 Thermal transition of PE-PilA fusion non His-tagged protein (LVL317) was compared with the thermal transition of both PE his-tagged (as described in Example 8) and cleaved PilA (as described in Example 8) proteins, purified as described above.

Before DSC, PE and PilA were dialyzed overnight in 10mM K2HPO4/KH2PO4 pH 6.5 + 0.04% Tween 80 (1:250 sample:buffer volume ratio) to have them in the same buffer as the fusion protein. After dialysis, proteins concentration was measured by BCA and adjusted to ml (PE) and 500ug/ml (PilA).

Analysis done on VPTM-DSC from MicroCal, LLC (part of GE Healthcare). The final dialysis buffer was used as reference and cted from the scans. DSC scan rate 90°C/hr. In order to evaluate the capacity to measure the thermal transition in the Final Container (FC) after formulation, the fusion protein was diluted to the FC concentration (60ug/ml). Final container data not shown.

Results: See Figure 17 for Thermal transition of PE-PilA fusion protein and PE and PilA proteins.

: PilA (1), n E (Prot E, PE) (2), PE-PilA PB not diluted 737ug/ml (3), and PE-PilA PB diluted at FC concentration 60ug/ml (4). 1 — PilA Tm: 53°C 2 — Protein E Tm: 63 3 — PE-PilA PB (Purified Bulk) not diluted 737ug/ml Tm1 : 537°C and Tm2: 661°C 4 — A PB diluted at FC concentration 60ug/ml Tm1: 532°C and Tm2: 67.6°C Two transitions were detected in the ed fusion protein (LVL317) (curves 3 and 4).

The Tm1 (537°C) of the PE-PilA fusion protein is r to PilA transition (53°C).

Significant shift of Tm2 in PE-PilA (66.1°C) as ed to PE transition (63°C). The fusion of both domains seems to ize the PE fragment.

WO 39225 The shift of Tm2 in the diluted fusion protein as compared to undiluted is a concentration artifact arising from the steep decreasing slope typical of aggregation which is tration dependant n g analysis of LVL735 and LVL778 were similar to that of LVL317.

Example 12: PE-PiIA fusion protein construct LVL291 anti-PiIA immunogenicity response in Balb/c mice.

The immune se directed t purified LVL291 PE-PilA fusion protein (the LVL291 fusion protein without the heterologous signal peptide) formulated in ASO3A was evaluated in Balb/c mice. Animals (20 mice/group) were immunized by the intramuscular route at days 0, 14 and 28 with 10 ug of PE (from vector pRlT16762), PilA (from vector pRlT16790) or PE- PilA, each formulated in ASO3A. The control group was vaccinated with ASO3A alone. Antibody response directed t each antigen was determined in individual sera collected at day 42.

No antibody response was obtained with the negative control. As shown in Figure 18, the antibody response directed against PilA was higher in mice immunized with the PE-PilA fusion compared to antibody response in mice immunized with monovalent PilA. The antibody responses directed against PE were similar in mice immunized with the fusion protein and mice immunized with monovalent PE. GMT = geometric means titer. Data were captured and analyzed with the SOFTMAX® Pro Software (Molecular Devices) running under WINDOWS® (Microsoft); the four ters logistic log function was used to calculate the standard curve.

The four-parameter logistic-log function describes, with a high degree of accuracy, the curve of the reference serum ying a pronounced sigmo'idal shape when plotted on an optical density-versus-concentration (log) scale. Antibody concentrations were calculated at each dilution of mice serum samples by interpolation of the rd curve. The dy in quality control sera and in n serum samples is obtained by averaging the values from all dilutions that fall within the working range (10-80 %) of the dilution curve of the reference.

Results are shown in Figure 18, which graphs the antibody responses against LVL291 PE- PilA fusion protein and t monovalent PE and PilA in the Balb/c mouse model. 2012/050236 Example 13: Murine nasopharzngeal colonization model. Immunization with PE-PiIA.

Challenge with NTHi strain 86-028NP and NTHi strain 3224A.

Balb/c female mice (20/group) were immunized intranasally at days 0 and 14 with 6pg of a purified PE-PiIA fusion protein (LVL291 for challenge with NP; LVL317 for challenge with strain 3224A) formulated with LT (heat labile toxin of Escheria coil) and on day 28 with 6 pg of a purified PE-PiIA fusion protein in phosphate buffered saline (PBS). Control mice (20/group) were vaccinated with LT alone. Mice were subsequently challenged intranasally with 5 x ‘106 CFU (colony forming units) of homologous NTHi strain 86-028NP and logous NTHi strain 3224A. Homology and heterology are ined by reference to the NTHi strain with which the mice were immunized. Bacterial colonies were counted in nasal cavities removed 1 and 2 days after the challenge. D1 = day 1. D2 = day 2.

PE-PiIA vaccination increased the clearance of NTHi strain NP and strain 3224A in the nasopharynx at day 1 and day 2 post challenge.

For the experiment performed with NTHi strain NP: A 2-way fixed ANOVA was performed using the log10 values of the counts as response, the fixed factors being the group (4 levels) and the day (2 levels). The assumption of variance heterogeneity was rejected and a model with heterogeneous variances was fitted to the data. No significant ction was detected between the 2 factors. The group fusion A (6 pg per mouse) significantly reduced CFU compared with the control group (LT); the geometric mean ratio being equal to 0.06 with a 95% confidence interval of 0.01, 0.25.

For the experiment conducted with NTHi strain 3224A: A 3-way fixed ANOVA was performed using the log10 values as response, the fixed factors being the group, the day, and the experiment. The Shapiro-Wilk and Levene’s test did not reject the assumptions of ity and of homogeneity of variances. No significant interaction between any of the 2 factors or between the 3 factors was detected and only main factors were kept in the analysis. PE-PiIA / LT significantly reduced CFU compared with the control group; the ric mean ratio being equal to 0.11 with a 95% confidence interval of 0.02, 0.61.

See Figure 19 for effect of PE-PiIA fusion protein vaccination on NTHi strain 86-028NP bacterial clearance in mouse nasopharynx.

See Figure 20 for effect of PE-PilA fusion protein ation on NTHi strain 3224A bacterial clearance in mouse nasopharynx.

Example 14: Murine nasophamngeal colonization model. zation with FHA.

Challenge with NTHi strain 3219C.

Female OF1 mice (20 mice/group) were immunized asally at days 0 and 14 with 3pg PilA (from vector 16790) formulated with LT and at day 28 with 3 ug PilA in PBS. Control mice were vaccinated with LT alone. Mice were subsequently challenged intranasally with 5 x ‘106 CFU of NTHi strain 3219C. Bacterial es were d in nasal cavities removed 3 and 4 days after the challenge. D3 = day 3. D4 = day 4.

See Figure 21 for effect of PilA vaccination on bacterial clearance in mouse nasopharynx.

Example 15: Murine nasopharyngeal colonization model. Immunization with PE.

Challenge with NTHi strain 3224A.

Balb/c female mice (20 mice/group) were immunized intranasally at days 0 and 14 with 3pg PE (from vector pRlT16762) formulated with LT and at day 28 with 3 ug PE in PBS. Control mice were vaccinated with LT alone. Mice were subsequently challenged intranasally with 5 x 106 CFU of NTHi strain 3224A. Bacterial colonies were counted in nasal cavities removed 3 and 4 days after the challenge. 10 mice were examined on day 3 (D3). 10 mice were examined on day 4 (D4). PE vaccination increased significantly the clearance of NTHi in the naso-pharynx at day 4 post challenge (Figure 22), using on the Dunn test for statistical analysis.

See Figure 22 for effect of PE vaccination on ial clearance in the nasopharynx of mice.

Example 16: Vibronectin binding. tion of Vibronectin binding by LVL317 & LVL735 PE-PiIA fusion n.

The ability of PE in the purified LVL317 PE-PilA fusion protein construct to bind to vitronectin was evaluated. Microtiter plates (POLYSORPTM, Nunc, Thermo Fisher Scientific) were coated with PE (from vector pRlT16762) or with purified LVL317 PE-PilA fusion protein (10 ug/ml).

Plates were washed four times with NaCl polysorbate 20, 0.05% (for example, TWEENTM 20) and d for one to two hours with PBS-BSA 1%. After four washings, vitronectin (Vitronectin from human plasma, SlGMA-ALDRICH®) was added (10 ug/ml), two fold diluted (12 dilutions), and the plates were incubated for 1h at room temperature. The plates were then washed 4 times with NaCl 150mM-polysorbate 20, 0.05% (for example TWEENTM 20) After washings, the bound vitronectin was detected using peroxydase sheep anti-human ectin (US Biological) followed by the on of ortho-phenylene diamine/H202 substrate. The color developed is directly proportional to the amount of antibody fixed to the vitronectin.

See Figure 23 for (a) LVL317 PE-PilA fusion protein bound to vitronectin. PilA = PilA from NTHi strain 86-028NP (as bed for pRlT16790); PE = Protein E (as described for pRlT16762) and (b) LVL317 and LVL735 PE-PilA fusion protein bound to vitronectin.

Example 17: Vibronectin binding. Inhibition of Vibronectin binding by antibodies directed against the LVL291 PE-PiIA fusion protein.

Microtiter plates (POLYSORPTM, Nunc, Thermo Fisher ific) were coated with PE (from vector pRlT16762) or with purified PE-PilA fusion protein (10 ug/ml). Plates were washed four times with NaCl 150mM-polysorbate 20, 0.05% (for example, TWEENTM 20) and blocked for two hours with A 1%. After gs, vitronectin (Vitronectin from human plasma, ALDRICH®) was added at 50ug/ml and purified antibodies anti-PE-PilA ced and purified in house) were two-fold serially diluted and incubated for 1h at room temperature. The plates were then washed 4 times with NaCl 150mM-polysorbate 20, 0.05% (for example, TWEENTM 20). After four washings, the bound vitronectin was detected using peroxydase sheep anti-Vitronectin (US Biological) followed by the addition of ortho-phenylene e/H202 ate. The color developed is directly proportional to the amount of antibody fixed to the vitronectin. tion of vitronectin binding to PE by polyclonal antibodies directed against PE-PilA was observed.

See Figure 24 for inhibition of vitronectin binding by polyclonal antibodies against PE-PilA fusion protein. e 18: Antigenicity of LVL291 PE-PiIA fusion protein. ELISA.

Purified LVL291 PE-PilA fusion protein was validated in an antigenicity test with lent proteins as control. The fusion protein was tested in a sandwich ELISA developed with polyclonal antibodies (rabbit and guinea pig) generated against the PE gene fragment coding for amino acids 22 to 160 of SEQ ID NO: 4 (as described for pRlT16711) or against PilA from NTHi strain 86-028NP (from vector pRlT16790).

PilA or PE was added at 100 ng/ml and serially two fold diluted. After 30 minutes incubation and after washing, the bound antigen was detected by a rabbit polyclonal serum obtained after immunisation with PE or PilA. The bound antibodies were detected using a peroxydase anti-rabbit lg on lmmunoResearch Laboratories, Inc.) followed by the addition of ortho- phenylene—diamine/HzOz substrate. The color developed is directly proportional to the amount of antigen t. Absorbance readings were measured using a spectrophotometer for microtiter plates. The antigenicity of the samples was ined by ison to the curve of the full length PE or full length PilA reference n and is expressed in ug/ml. The reference represented 100% of antigenicity.

As observed in the Table 6: Antigenicity was observed with the purified LVL291 PE-PilA fusion protein compared to the monovalent PE and PilA antigens.

Table 6 : Relative antigenicity obtained with purified LVL291 PE-PilA fusion protein in the antigenicity test.

—PErelative antigenicity (%) Protein E as Reference 100 PE-PilA 130-148 —PErelative antigenicity (%) PilA as Reference 100 PE-PilA 120-152 Example 19: Immunogenicity of LVL735 PE-PiIA fusion protein.

Female Balb/c mice (n = 34) were immunized by the uscular route at days 0, 14 and 28 with 50 pl of vaccine formulation containing 1, 0.2 or 0.04 pg of A fusion protein LVL317 or LVL735 formulated within ASO‘IE or AIPO4 (aluminium phosphate). The antibody responses to 2012/050236 PE and PilA were determined in individual sera collected at day 42 and the lgG level against PE and PilA was measured and expressed in ug /m|.

See Figure 27 for PE and PilA antibody response to LVL317 and LVL735. GMC= geometric mean concentration = geometric means titer.

. GMT IC = confidence als.

Example 20: Protective efficacy of the LVL735 and LVL317 fusion proteins in a mouse model of Non-tzgeable hilus influenzae nasophamngeal colonization.

Female Balb/c mice were intranasally immunized at days 0 and 14 with 10 ul of vaccine formulation containing 5.8 ug of LVL735 or LVL317 admixed with 0.5 ug of E. coli labile toxin (LT). A r dose of 5.8 ug of non-adjuvanted LVL735 or LVL317 was administered at day 28. Control mice were vaccinated with LT alone at days 0 and 14, and PBS at day 28. Animals were intranasally challenged with 5 x ‘106 cfu of NTHi 3224A strain at day 42. Bacterial colonies were counted in nasal cavities removed 1 and 2 days after the challenge (n = 10/time-point).

Nasal cavities are homogenized in medium and a bacterial quantification is performed. Results are well expressed in CFU/ml.

See Figure 28 for the effect of LVL735 and LVL317 vaccination on bacterial nce in a mouse model of non-typeable Haemophi/us zae nasopharyngeal colonization.

Claims (47)

1. CLAIMS 1. A fusion protein of formula I: (X) m – (R1)n – A – (Y) o – B – (Z)p (formula I) wherein: X is a signal peptide or MHHHHHH (SEQ ID NO. 2); m is 0 or 1; R1 is an amino acid; n is 0, 1, 2, 3, 4, 5 or 6; A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, or PilA from Haemophilus influenzae or an immunogenic fragment thereof; Y is selected from the group consisting of GG, SG, SS, GGG and (G)h n h is 4, 5, 6, 7, 8, 9, or 10; o is 0 or 1; B is PilA from Haemophilus influenzae or an immunogenic fragment thereof, or Protein E from Haemophilus nzae or an immunogenic fragment thereof; Z is HH (SEQ ID NO. 3); and p is 0 or 1, wherein when A is Protein E from Haemophilus influenzae or an immunogenic fragment thereof, B is not Protein E from Haemophilus influenzae or an immunogenic fragment thereof; and wherein when A is PilA from Haemophilus influenzae or an genic fragment thereof, B is not PilA from Haemophilus influenzae or an immunogenic fragment thereof.
2. 2. A fusion n according to claim 1 wherein X is selected from the group consisting of FlgI, NadA and pelB.
3. 3. A fusion protein according to any of claims 1-2 n m is 0.
4. 4. A fusion protein according to any of claims 1-3 wherein n is 0.
5. 5. A fusion protein according to any of claims 1-4 wherein A is an immunogenic nt of Protein E, wherein Protein E is selected from any one of SEQ ID NO. 4 – SEQ ID NO. 57.
6. 6. A fusion protein according to any of claims 1-5 n A is the genic fragment of Protein E from H. nzae as set forth in SEQ ID NO: 124.
7. 7. A fusion protein according to any of claims 1-6 wherein Y is GG.
8. 8. A fusion protein according to any one of claims 1-7 wherein B is an immunogenic fragment of PilA, wherein PilA is selected from any one of SEQ ID NO. 58 – SEQ ID NO. 121.
9. 9. A fusion protein according to any of claims 1-8 wherein B is the immunogenic fragment of PilA from H. influenzae as set forth in SEQ ID NO. 127.
10. 10. A fusion protein selected from the group consisting of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO.182, SEQ ID NO.184, SEQ ID NO.186, SEQ ID NO.188, SEQ ID NO. 190, SEQ ID NO.192, SEQ ID NO.194, SEQ ID NO.196, SEQ ID NO.198, SEQ ID NO.200, SEQ ID NO.202 and SEQ ID NO.204.
11. 11. A fusion protein approximately 95% cal to any of SEQ ID NO. 136, SEQ ID NO. 138, SEQ ID NO. 140, SEQ ID NO. 142, SEQ ID NO. 144, SEQ ID NO. 146, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 182, SEQ ID NO. 184, SEQ ID NO. 186, SEQ ID NO. 186, SEQ ID NO. 188, SEQ ID NO. 190, SEQ ID NO. 192, SEQ ID NO. 194, SEQ ID NO. 196, SEQ ID NO. 198, SEQ ID NO. 200, SEQ ID NO. 202 or SEQ ID NO. 204.
12. 12. A fusion protein of claim 10 or claim 11 wherein the signal peptide has been removed.
13. 13. The fusion protein of SEQ ID NO. 148, wherein the signal peptide has been d, SEQ ID NO. 177 (QIQKAEQN DVKLAPPTDV RSGYIRLVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK EPKR YARSVRQYKI LNCANYHLTQ DEFW GQGLRAAPKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA PYKADVELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQ).
14. 14. The fusion protein of SEQ ID NO. 194, wherein the signal peptide has been removed, SEQ ID NO. 219 (IQKAEQND VKLAPPTDVR SGYIRLVKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV EFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG VTWTTTCKGT DASLFPANFC GSVTQ).
15. 15. An immunogenic composition comprising isolated Protein E from H. influenzae and isolated PilA from H. influenzae.
16. 16. An immunogenic composition of claim 15 wherein Protein E is a polypeptide of SEQ ID NO. 4, a polypeptide comprising a sequence having at least 75%, 77%, 80%, 85%, 90%, 95%, 97%, 99% or 100% ty, over the entire length, to SEQ ID NO. 4, or is a polypeptide sing an genic fragment of at least 7, 10, 15, 20, 25, 30 or 50 uous amino acids of SEQ ID NO. 4.
17. 17. The immunogenic composition of claim 16, wherein the immunogenic fragment comprises a B and/or T cell epitope of SEQ ID NO: 4.
18. 18. The genic composition of claims 15-17 wherein Protein E is capable of eliciting an immune response which recognizes SEQ ID NO. 4.
19. 19. An immunogenic composition of claims 15-18 n PilA is a ptide of SEQ ID NO. 58, a polypeptide comprising a sequence having at least 80%, 85%, 90%, 95%, 97% or 100% identity, over the entire length, to SEQ ID NO. 58, or is a polypeptide comprising an immunogenic fragment of at least 7, 10, 15, 20, 25, 30 or 50 contiguous amino acids of SEQ ID NO. 58.
20. 20. The immunogenic composition of claim 19, n the immunogenic fragment comprises a B and/or T cell epitope of SEQ ID NO: 58.
21. 21. An immunogenic composition of any of claims 15-20 wherein PilA is capable of eliciting an immune response which recognizes SEQ ID NO. 58.
22. 22. The immunogenic composition of claims 15-21, wherein the Protein E from H. influenzae and the PilA from H. influenzae are comprised as the fusion protein of claims 1-14.
23. 23. An immunogenic ition comprising the fusion protein of SEQ ID NO. 177 (QIQKAEQN DVKLAPPTDV LVKN VNYYIDSESI WVDNQEPQIV HFDAVVNLDK GLYVYPEPKR YARSVRQYKI LNCANYHLTQ VRTDFYDEFW APKK QKKHTLSLTP DTTLYNAAQI ICANYGEAFS VDKKGGTKKA AVSELLQASA ELCV YSTNETTNCT GGKNGIAADI TTAKGYVKSV TTSNGAITVK GDGTLANMEY ILQATGNAAT GVTWTTTCKG TDASLFPANF CGSVTQ).
24. 24. An genic composition comprising the fusion protein of SEQ ID NO. 219 (IQKAEQND VKLAPPTDVR VKNV NYYIDSESIW VDNQEPQIVH FDAVVNLDKG LYVYPEPKRY ARSVRQYKIL NCANYHLTQV RTDFYDEFWG QGLRAAPKKQ KKHTLSLTPD TTLYNAAQII CANYGEAFSV DKKGGTKKAA VSELLQASAP YKADVELCVY STNETTNCTG GKNGIAADIT TAKGYVKSVT TSNGAITVKG DGTLANMEYI LQATGNAATG CKGT DASLFPANFC .
25. 25. A vaccine comprising the fusion protein of any of claims 1-14 or genic compositions of any of claims 15-24.
26. 26. The use of a fusion protein according to any one of claims 1 to 14, an immunogenic composition according to any of claims 15-24 or the vaccine of claim 25, in the manufacture of a medicament for the treatment or prevention of otitis media in a subject in need thereof.
27. 27. The use according to claim 26, wherein the immunogenic composition is a composition according to claim 23 or claim 24.
28. 28. The use of a fusion protein according to any one of claims 1 to 14, an immunogenic composition according to any of claims 15-24 or the vaccine of claim 25, in the manufacture of a medicament for the treatment or tion of acute exacerbations of chronic obstructive pulmonary disease (AECOPD) in a subject in need thereof.
29. 29. The use according to claim 28, wherein the genic composition is a composition according to claim 23 or claim 24.
30. 30. The use of a fusion protein according to any one of claims 1 to 14, an genic composition according to any of claims 15-24 or the vaccine of claim 25 in the manufacture of a medicament for the treatment or prevention of pneumonia in a subject in need thereof.
31. 31. The use according to claim 30, wherein the immunogenic composition is a composition ing to claim 23 or claim 24.
32. 32. The use of a fusion protein according to any one of claims 1 to 14, an immunogenic composition according to any of claims 15-24 or the vaccine of claim 25 in the manufacture of a medicament for the treatment or tion of a H. influenzae infection or disease.
33. 33. The use according to claim 32, wherein the immunogenic composition is a composition according to claim 20 or claim 21.
34. 34. The use of claim 32 or claim 33, wherein the H. nzae infection or disease is an NTHi infection or disease.
35. 35. The fusion protein of claims 1-14, or the immunogenic composition of claims 15-24, or the vaccine of claim 25, for use in the treatment or prevention of otitis media.
36. 36. The fusion protein, the immunogenic ition, or the vaccine of claims 1-25, 35, for use in the treatment or prevention of acute bations of chronic obstructive pulmonary disease D).
37. 37. The fusion protein, the immunogenic composition, or the vaccine of claims 1-25, 35-36, for use in the treatment or prevention of pneumonia.
38. 38. The fusion protein, the immunogenic composition, or the vaccine of claims 1-25, 35-37, for use in the treatment or prevention of H. influenzae infection or disease.
39. 39. The fusion protein, the immunogenic composition, or the vaccine of claim 38, for use in the treatment or prevention of NTHi ion or disease.
40. 40. A process for producing periplasmic expression of a fusion protein of any one of claims 1 to 14, wherein the process comprises inducing expression of proteins containing a signal peptide.
41. 41. The process of claim 40 wherein the signal peptide is from FlgI.
42. 42. The process of claim 40 wherein the signal e is from pelB.
43. 43. A process for making a vaccine comprising the process of any one of claims 40-42.
44. 44. A fusion protein according to any one of claims 1 to 14, substantially as herein bed or exemplified.
45. 45. An immunogenic composition according to any one of claims 15 to 24, ntially as herein described or exemplified.
46. 46. A vaccine according to claim 25, substantially as herein described or exemplified.
47. 47. A use according to any one of claims 26-34, substantially as herein described or ified. 51. A process according to claim 40, substantially as herein described or exemplified.